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Genetic variants in the triggering receptor bidding on myeloid beef 2 (TREM2) accept been affiliated to Nasu-Hakola disease, Alzheimer’s ache (AD), Parkinson’s disease, amyotrophic crabbed sclerosis, frontotemporal dementia (FTD), and FTD-like affection afterwards cartilage involvement. TREM2 is an congenital allowed receptor preferentially bidding by microglia and is complex in deepening and phagocytosis. Whether and how TREM2 missense mutations affect TREM2 action is unclear. We address that missense mutations associated with FTD and FTD-like affection abate TREM2 maturation, abate address by ADAM proteases, and blemish the phagocytic action of TREM2-expressing cells. As a aftereffect of bargain shedding, TREM2 is about absent in the cerebrospinal aqueous (CSF) and claret of a accommodating with FTD-like syndrome. A abatement in acrid TREM2 was additionally empiric in the CSF of patients with AD and FTD, added suggesting that bargain TREM2 action may accord to added accident for two neurodegenerative disorders.
Homozygous loss-of-function mutations in the triggering receptor bidding on myeloid beef 2 (TREM2) gene account polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy [also accustomed as Nasu-Hakola ache (NHD)], a ache characterized by abate swellings and common cartilage fractures (1). During ache progression, NHD patients advance acoustic syndromes evocative of the behavioral accession of frontotemporal dementia (FTD) (1). Recently, homozygous missense mutations of TREM2, such as the p.T66M and the p.Y38C mutations, as able-bodied as a admixture heterozygous missense alteration accept been articular to account an FTD-like affection afterwards cartilage anatomy (2, 3). Abiogenetic screenings accept now additionally articular heterozygous missense mutations in TREM2 as accident factors for Alzheimer’s ache (AD), Parkinson’s ache (PD), amyotrophic crabbed sclerosis (ALS), and FTD (4–9). Best appear missense mutations are amid in the ectodomain of TREM2, a membrane-bound blazon 1 protein (Fig. 1A). Integrated arrangement appraisal adapted a axial role for TREM2 in assorted academician areas (10), breadth it is mainly bidding in microglia beef acclimation capital functions including phagocytosis and the abatement of apoptotic neurons (11–14).
(A) Illustration of membrane-bound TREM2. Aloft address by ADAM10, the actual C-terminal butt of TREM2 is broken aural the film by γ-secretase (17). Ig, immunoglobulin; HA, hemagglutinin; TM, transmembrane domain; ICD, intracellular domain. (B) Pharmacological inhibition of ADAM proteases appliance a ample ADAM inhibitor (GM 6001) or a added careful ADAM10 inhibitor (GI 254023X) but not a added careful ADAM17 (GL 506-3) or BACE1 (C1) inhibitor bargain sTREM2 bearing and counterbalanced absolutely glycosylated, corpuscle surface–associated, complete membrane-bound wild-type (WT) TREM2. (C) Baby interfering RNA (siRNA)–mediated ADAM10 altercation accustomed bargain ADAM10-mediated TREM2 shedding. Hashtags announce all-embracing immunoreactivity. Anti-calnexin antibiotic (B and C) was acclimated as a loading control. Bar graphs (B and C) appearance enzyme-linked immunosorbent appraisal (ELISA) altitude of sTREM2 about to control-treated cells. Quantitative abstracts are represented as agency ± SD from at atomic two absolute experiments; n = 4 to 7 (B) and n = 8 (C). Statistical differences were afflicted by Mann-Whitney U test. **P < 0.01.
The TREM2 homolog TREM1 is begin as a acrid accession in the serum of patients with catchbasin shock and is buried from monocytes aloft dispatch with lipopolysaccharide (15). Furthermore, TREM2 was additionally empiric in claret and cerebrospinal aqueous (CSF) samples from patients with assorted sclerosis (16). In band with these findings, contempo affirmation suggests that TREM2 is a substrate for adapted intramembrane proteolysis (RIP) (17). RIP substrates are membrane-bound proteins, whose ectodomains are appear aloft address by proteases such as associates of the ADAM (a disintegrin and metalloproteinase domain-containing protein) or BACE (β-site APP cleaving enzyme) ancestors (18). Aloft abatement of the ectodomain, the actual membrane-retained butt is added candy by intramembrane proteolysis (19).
Whether and how missense mutations affect TREM2 action is elusive. Here, we advised whether TREM2 missense mutations begin in patients with FTD, FTD-like affection afterwards cartilage involvement, AD, and added neurodegenerative diseases affect the carriage and processing of the TREM2 protein and appropriately may account its accident of function.
Soluble bits of TREM2 (sTREM2) accept been empiric in supernatants of blooming corpuscle cultures as able-bodied as in claret and CSF samples from patients with noninflammatory acoustic diseases and assorted sclerosis (16). Constant with that, we empiric buried bits of TREM2 (Fig. 1B) brief as assorted bands in the ambit of 36 to 50 kD as able-bodied as the feature membrane-bound TREM2 brief amid 36 and 60 kD (Fig. 1B) in isogenic animal beginning branch 293 beef (HEK293 Flp-In) durably cogent a distinct archetype of animal wild-type TREM2. We did not acquisition C-terminal FLAG epitope absolute bits in the conditioned medium, implying that sTREM2 is produced by ectodomain address (fig. S1). Moreover, in HEK293 Flp-In cells, the ample ADAM inhibitor GM 6001 bargain beard of sTREM2 as did the added careful ADAM10 inhibitor GI 254023X, but not the ADAM17-specific inhibitor GL 506-3 or the BACE inhibitor C3 (Fig. 1B). Bargain sTREM2 activated with an access in absolutely glycosylated complete membrane-bound TREM2 (Fig. 1B, average panel; for added appraisal of adolescent and complete TREM2, see the pulse-chase agreement in Fig. 2E and the deglycosylation abstracts in fig. S2). siRNA-mediated altercation appear ADAM10 as a aloft sheddase of TREM2 in HEK293 Flp-In beef (Fig. 1C).
(A) Anti-TREM2 and anti-HA immunoblotting of sTREM2 in supernatants from beef cogent the FTD- and FTD-like–associated TREM2 mutations p.T66M and p.Y38C or mutations of conserved cysteine residues at positions 36 and 60. Lower console shows altitude of sTREM2 by ELISA on supernatants from abiding HEK293 Flp-In cells. (B) Adolescent aberrant TREM2 (black arrowhead) accumulated, admitting the complete anatomy and CTFs were bargain compared to WT TREM2. An accession proteolytic accident appears to aftermath accessory amounts of a beyond CTF (gray arrowhead). (C) Altitude of immunoblots shows a baby access in adolescent p.R47H TREM2 and a cogent abatement in the arrangement of mature-to-immature TREM2. (D) Coexpression of DAP12, the signaling adaptor of TREM2, did not affect bargain maturation and beard of aberrant TREM2. (E) Pulse-chase abstracts appear a best half-life for aberrant TREM2 aural corpuscle lysates accompanied by a basal absolution of sTREM2 into the medium. (F) Apparent biotinylation of complete surface-exposed aberrant TREM2. Anti-APP (A) or anti-calnexin (B) antibodies were acclimated as loading controls for supernatants or film fractions, respectively. Anti-calnexin antibiotic was acclimated to prove careful corpuscle apparent labeling. NT, nontransfected HEK293 Flp-In host corpuscle line. Quantitative abstracts are represented as agency ± SD from three absolute abstracts appliance three absolute corpuscle ambit for WT or p.R47H TREM2. n > 6 (A) and n = 9 (C). Statistical differences were afflicted by Mann-Whitney U test. **P < 0.01; ***P < 0.001.
Expression of the FTD and FTD-like TREM2 p.T66M and p.Y38C mutations (2, 9) appear a able abridgement of sTREM2 in conditioned average from isogenic HEK293 Flp-In beef (Fig. 2A). Furthermore, the TREM2 p.R47H mutation, which increases the accident for AD, PD, and ALS (4–7), additionally showed bargain beard of sTREM2 admitting to a lower admeasurement (Fig. 2A). In parallel, we empiric an accession of membrane-bound adolescent feature TREM2 p.T66M and p.Y38C calm with a abridgement in TREM2 C-terminal bits (CTFs) generated by ADAM10-mediated proteolytic break (Fig. 2B). The apprehension of accumulating amounts of adolescent aberrant TREM2 variants additionally accustomed that the mutations do not abate antibiotic recognition. Constant with a weaker aftereffect of the p.R47H alteration on TREM2 shedding, we empiric a abundant beneath apparent accession of adolescent p.R47H aberrant TREM2 (Fig. 2B; quantitated in Fig. 2C). Mutagenesis of conserved cysteine residues (p.C36A and p.C60A) blocked TREM2 address like the p.T66M and p.Y38C mutations (Fig. 2A) and acquired an accession of adolescent membrane-bound feature TREM2 as able-bodied as a abridgement of the CTF (Fig. 2B). This added supports the achievability that aberrant TREM2 is misfolded and retained aural the cell. Coexpression of DAP12, the signaling adaptor for TREM2 (20), which is not bidding in HEK293 Flp-In cells, did not restore carriage and address of aberrant TREM2 (Fig. 2D). Pulse-chase abstracts showed that wild-type membrane-bound feature TREM2 accomplished from a low–molecular weight adolescent breed to a higher–molecular weight complete breed best acceptable by glycosylation (Fig. 2E and fig. S2). Aloft maturation afterwards 60 to 90 min of algid chase, TREM2 was afford and accumulated over time in the conditioned average (Fig. 2E). In adverse to wild-type TREM2, aberrant p.T66M TREM2 bootless to complete efficiently, constant in the accession of adolescent TREM2 in the corpuscle lysate and inefficient bearing of sTREM2 (Fig. 2E). Accordingly, corpuscle apparent TREM2 was decreased aloft announcement of the TREM2 p.T66M, p.Y38C, p.C36A, and p.C60A mutants compared to wild-type TREM2 (Fig. 2F). Constant with beneath apparent furnishings of the p.R47H alteration on TREM2 maturation and sTREM2 generation, p.R47H TREM2 showed agnate corpuscle apparent announcement compared to wild-type TREM2 (Fig. 2F). Immunohistochemistry additionally showed acutely bargain corpuscle apparent announcement of TREM2 p.T66M, p.Y38C, p.C36A, and p.C60A accompanied by an access of intracellular staining, which colocalized preferentially with the endoplasmic cloth (ER) brand calnexin (Fig. 3A). Constant with our biochemical findings, the p.R47H TREM2 alteration showed beneath intracellular accession (Fig. 3A), and consequently, able-bodied announcement of corpuscle apparent TREM2 was detected (Fig. 3B). Thus, mutations associated with FTD and FTD-like affection afflicted TREM2 maturation, corpuscle apparent transport, and proteolytic processing, admitting the AD-associated p.R47H alteration had a balmy aftereffect on maturation and secretion, which is at the absolute of biochemical detection.
(A) Double immunofluorescence of TREM2 labeled with anti-HA antibiotic and anti-calnexin antibody. White boxes on the alloyed images characterize the continued breadth apparent in the images to the right. White arrows in continued images highlight called areas assuming TREM2 and calnexin colocalization. (B) Apparent staining of WT and aberrant TREM2-expressing HEK293 Flp-In cells. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole.
Within the animal brain, TREM2 is primarily bidding in microglia beef (11–14). Therefore, we aimed to affirm our allegation in an in vivo accordant setting. To do so, we bidding wild-type TREM2, as able-bodied as the TREM2 mutations p.T66M, p.Y38C, p.R47H, and p.C36A, in the murine microglial BV2 corpuscle line, a archetypal frequently acclimated for in vitro studies of microglial action (21–23). Reverse archetype polymerase alternation acknowledgment (RT-PCR) appraisal accustomed the announcement of called microglial markers CD11b and CD68 as able-bodied as the announcement of Trem2 and Dap12 in the BV2 corpuscle band (Fig. 4A). The p.T66M, p.Y38C, and p.C36A mutations apparent bargain address of sTREM2 in BV2 beef (Fig. 4B), as empiric in HEK293 Flp-In beef (Fig. 2A). Remarkably, the TREM2 p.R47H aberrant consistently showed bargain announcement in the film atom of briefly transfected BV2 beef (Fig. 4B), an ascertainment that was additionally accustomed in durably transfected BV2 corpuscle ambit (fig. S3). Nevertheless, agnate to our allegation in HEK293 Flp-In beef (Fig. 2A), the p.R47H alteration showed bargain beard of sTREM2 (Fig. 4B). Address of TREM2 by microglia beef was additionally advised by proteases of the ADAM ancestors but not BACE1 (Fig. 4C). In the microglia BV2 corpuscle line, both ADAM10 and ADAM17 contributed to address of TREM2 (Fig. 4C). We additionally accustomed bargain corpuscle apparent carriage of aberrant TREM2 in BV2 beef by investigating wild-type and p.T66M aberrant TREM2 in a corpuscle apparent biotinylation assay. In band with our allegation in HEK293 Flp-In cells, we begin beneath corpuscle apparent TREM2 aloft announcement of the p.T66M alteration (Fig. 4D). Moreover, immunohistochemistry additionally absolutely accustomed the assimilation of aberrant TREM2 in BV2 beef (Fig. 4E). Although wild-type TREM2 was predominantly empiric in the Golgi, the p.T66M, p.Y38C, and p.C36A aberrant variants were retained predominantly aural the ER (Fig. 4E). Constant with bargain furnishings of the p.R47H alteration on maturation of TREM2 and sTREM2 generation, this accession was predominantly amid aural the Golgi (Fig. 4E). Thus, the furnishings of TREM2 mutations on bargain maturation and corpuscle apparent carriage as able-bodied as on proteolytic processing were accustomed in the microglial BV2 corpuscle line.
(A) RT-PCR appraisal of Trem2, Dap12, and microglial markers (CD11b and CD68) in BV2 cells. (B) Comparison of TREM2 processing in BV2 beef briefly cogent WT or aberrant TREM2. Note that announcement of the p.R47H accession was lower in all conducted experiments. Altitude of sTREM2 by ELISA normalized to the announcement of adolescent TREM2 (lower panel). The AD accession p.R47H additionally showed bargain sTREM2 generation, but to a bottom admeasurement than the added advised mutants. Hashtag indicates a protein that cross-reacts with the animal anti-TREM2 antibody. (C) Agnate to the case with HEK293 Flp-In beef (see Fig. 1), pharmacologic inhibition of ADAM proteases with GM 6001 (broad ADAM inhibitor), GI 254023X (ADAM10 inhibitor), and GL 506-3 (ADAM17 inhibitor) bargain the bearing of sTREM2 in the microglial BV2 corpuscle line. Anti-calnexin antibiotic was acclimated as a loading ascendancy for film fractions in (B) and (C). (D) Apparent biotinylation of complete surface-exposed aberrant TREM2. (E) Double immunofluorescence of TREM2 labeled with anti-HA antibiotic and anti-calnexin antibody. White boxes on the alloyed images characterize the breadth continued in the images to the right. White arrows in continued images highlight called areas with TREM2 and calnexin colocalization. Scale bars, 20 μm. Quantitative abstracts are represented as agency ± SD from at atomic two absolute experiments; n = 4. Statistical differences were afflicted by Mann-Whitney U test. *P < 0.05; n.s., nonsignificant.
To access affirmation for bargain corpuscle apparent carriage of aberrant TREM2 in patients with FTD-like affection afterwards cartilage involvement, we analyzed sTREM2 concentrations in the CSF and claret of a accommodating accustomed the TREM2 p.T66M alteration (2) as able-bodied as accession accommodating with the p.Q33X alteration (2) for which alone claret was available. We accustomed a awful acute sTREM2 ELISA (fig. S4, A to D) that showed acceptable alternation with a ahead appear sTREM2 ELISA (16), which acclimated absolute antibodies (fig. S4B; Spearman rho = 0.521, P < 0.001). Constant with the tissue ability analysis, these two absolute ELISAs as able-bodied as immunoblotting appear the basic absence of sTREM2 in CSF from a accommodating with a homozygous TREM2 p.T66M alteration (Fig. 5A and fig. S4, E and F). Furthermore, the claret absorption of sTREM2 in this accommodating was additionally beneath the apprehension absolute and that of the accommodating accustomed the p.Q33X alteration was actual low (Fig. 5B). Because TREM2 is genetically affiliated not alone to NHD and FTD-like affection afterwards cartilage captivation but additionally to AD (4, 5) and FTD (6, 8, 9), we analyzed sTREM2 concentrations in CSF samples of a set of well-characterized FTD and AD patients (Table 1) and compared them with those of neurologically accustomed controls. Although we empiric an overlap amid both groups, statistical appraisal appear a cogent abridgement of sTREM2 in AD and FTD patients compared to ascendancy individuals (Table 1 and Fig. 5A; P = 0.001 and P < 0.001, respectively, Mann-Whitney U test). The cogent abatement in sTREM2 concentrations in CSF in AD and FTD patients compared to controls was still present afterwards authoritative for the aftereffect of gender and age, and was absolute of the clinics accession CSF [P = 0.001 and P < 0.001, respectively, appraisal of covariance (ANCOVA)]. In adverse to CSF measurements, we did not ascertain any aberration in sTREM2 concentrations in claret amid the controls and the AD and FTD patients (Table 2 and Fig. 5B; P = 0.872, Kruskal-Wallis).
(A) ELISA-based appraisal of sTREM2 in CSF samples shows basic absence of sTREM2 in the TREM2 p.T66M alteration carrier, admitting able-bodied concentrations of sTREM2 were detected in all ascendancy samples (n = 88). A abridgement in sTREM2 was empiric in AD patients (n = 56; Pcontrol vs. AD = 0.001) and FTD patients (n = 50; Pcontrol vs. FTD < 0.001). Horizontal confined announce average sTREM2 concentrations per accumulation with the interquartile ambit (IQR). (B) sTREM2 concentrations in claret (nControl = 86; nAD = 51; nFTD = 35) showed no cogent aberration amid these groups. Claret from the homozygous TREM2 p.T66M alteration carrier was the alone sample (1 of 174 samples) with ephemeral sTREM2. Additionally, claret from a homozygous TREM2 p.Q33X alteration carrier (2) additionally showed one of the everyman sTREM2 concentrations of all abstinent samples.
Data are bidding as cardinal of patients (percent), beggarly ± SD, or average (IQR) as appropriate. Probability belief (P) denote differences amid control, AD, and FTD accommodating groups. χ2 tests were acclimated for gender. One-way appraisal of about-face (ANOVA) was acclimated to analyze age amid groups followed by Tukey cavalcade hoc test. CSF biomarkers and sTREM2 were evaluated by nonparametric statistical appraisal (Kruskal-Wallis and cavalcade hoc with Mann-Whitney U test).
Data are bidding as cardinal of patients (percent), beggarly ± SD, or average (IQR) as appropriate. Probability belief (P) denote differences amid control, AD, and FTD accommodating groups. χ2 tests were acclimated for gender. One-way ANOVA was acclimated to analyze age amid groups followed by Tukey cavalcade hoc test. CSF biomarkers and sTREM2 were evaluated by nonparametric statistical appraisal (Kruskal-Wallis and cavalcade hoc with Mann-Whitney U test).
Reduction of corpuscle apparent TREM2 in microglia cells, the corpuscle blazon in which TREM2 is selectively bidding aural the brain, was appear to abate abatement of cellular bits and apoptotic neurons (14). In band with this, primary microglia from Trem2 knockout mice (Trem2−/−) (24), which do not aftermath apparent sTrem2 in conditioned average (Fig. 6A), showed a bargain phagocytic accommodation compared to wild-type controls in an appraisal appliance Escherichia coli conjugated to pHrodo that alone yields a beaming arresting in an acerb alcove (Fig. 6, B and C). Although the all-embracing accommodation of Trem2−/− microglia to phagocytose E. coli was alone hardly reduced, Trem2−/− microglia seemed to be beneath competent in phagocytosing a beyond bulk of bacilli (Fig. 6, B and C).
(A) ELISA-based appraisal of sTrem2 in the supernatants of WT or Trem2 knockout (Trem2−/−) primary microglia. (B and C) Breeze cytometric appraisal of the phagocytic accommodation of primary microglia appliance pHrodo E. coli as ambition particles. Q2, 50th percentile; Q3, 75th percentile. (D) Illustration of TREM2-DAP12 admixture assemble (25) acclimated in the phagocytosis assays apparent in (F), (G), and (I). ITAM, immunoreceptor tyrosine-based activation motif. (E) Anti-TREM2 immunoblot of TREM2-DAP12–expressing HEK293 Flp-In cells. Anti-calnexin antibiotic was acclimated as a loading control. (F) Phagocytosis of fluorescently labeled acrylic chaplet in HEK293 Flp-In beef durably cogent TREM2-DAP12 admixture constructs. Phagocytic basis (percentage of beef that phagocytose chaplet bidding about to WT) from three absolute abstracts is apparent as beggarly ± SD. (G) Phagocytosis of pHrodo E. coli in HEK293 Flp-In beef durably cogent TREM2-DAP12 admixture constructs and quantified by breeze cytometry. Abstracts are depicted as agency ± SD from at atomic two absolute experiments. (H to J) Phagocytosis of 6-carboxyfluorescein (FAM)–labeled Aβ1–42 by (H) BV2 beef durably cogent WT or aberrant TREM2, (I) HEK293 Flp-In beef durably cogent TREM2-DAP12 admixture constructs, and (J) primary microglia acquired from Trem2−/− mice is apparent as beggarly ± SD from two absolute abstracts (J) or three absolute abstracts (H and I) and is bidding about to WT controls. In all assays, cytochalasin D (10 μM) was acclimated as a abrogating ascendancy to arrest phagocytosis. Statistical differences were afflicted by Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P ≤ 0.001.
Because our appraisal in both the BV2 microglial corpuscle band and the HEK293 Flp-In beef (Figs. 2 to 4) appear bargain carriage of aberrant TREM2 to the corpuscle surface, we abutting advised whether this would associate with a bargain accommodation for phagocytosis. Constant with that, bean uptake assays appliance HEK293 Flp-In beef cogent the ectodomain of TREM2 alloyed to the transmembrane and signaling breadth of DAP12 (25, 26) (Fig. 6, D and E) accustomed that bargain corpuscle apparent localization and beard of aberrant TREM2 (p.T66M and p.Y38C) anon activated with a bargain phagocytic accommodation appliance opsonized beaming acrylic chaplet (Fig. 6F; **P = 0.004, Mann-Whitney U test). Moreover, an absolute appraisal appliance E. coli conjugated to pHrodo produced agnate after-effects (Fig. 6G and fig. S5). In band with our biochemical abstracts in BV2 microglial beef as able-bodied as in HEK293 Flp-In cells, the AD accident accession p.R47H broken phagocytosis to a abundant bottom admeasurement in the bean uptake appraisal (Fig. 6F; **P = 0.004, Mann-Whitney U test) and was alike accustomed for uptake of bacilli (Fig. 6G; n.s., P = 0.191, Mann-Whitney U test). Finally, we acclimated the pathologically accordant amyloid β-peptide 1–42 (Aβ1–42) as a substrate in the phagocytosis assays and accustomed that TREM2 accident of action due to the missense mutations decidedly broken phagocytosis of Aβ1–42 in three absolute corpuscle ambit including BV2 microglial beef (Fig. 6H; ***P < 0.001, Mann-Whitney U test), HEK293 Flp-In beef (Fig. 6I; **P < 0.01, ***P < 0.001, Mann-Whitney U test), and Trem2−/− primary microglia (Fig. 6J; *P = 0.016, **P < 0.01, Mann-Whitney U test). Thus, mutations that acutely abate maturation of TREM2, such as p.T66M and p.Y38C, aftereffect in a cogent accident of action of TREM2 as abstinent by three absolute phagocytosis assays.
These allegation advance that announcement of complete membrane-bound TREM2 correlates with phagocytic action of microglia. Therefore, we articular that TREM2 may be articulate to access approval of cellular bits and amyloidogenic seeds. As affidavit of assumption and as an added articulation amid corpuscle apparent TREM2 announcement and phagocytosis, we advised whether inhibition of TREM2 shedding, which increases corpuscle apparent TREM2 (Fig. 1, B and C), increases phagocytosis. In band with the abstracts in Fig. 1, aloft appraisal of BV2 microglial beef endogenously cogent Trem2 (Fig. 4A), with a ample pharmacological inhibitor of ADAM proteases (GM 6001), proteolytic processing of autogenous Trem2 was decidedly bargain (Fig. 7A; *P = 0.019; n.s., P > 0.05, Mann-Whitney U test). Moreover, bargain Trem2 address activated with a cogent access in phagocytosis as apparent by added uptake of E. coli (Fig. 7B; ***P < 0.001; n.s., P > 0.05, Mann-Whitney U test). Thus, these allegation accommodate added affirmation for a articulation amid corpuscle apparent announcement of Trem2 and phagocytosis.
(A) Autogenous sTrem2 in supernatants from BV2 beef afterwards inhibition of ADAM proteases (GM 6001) or BACE1 (C3). (B) Phagocytosis of pHrodo E. coli in BV2 beef advised with GM 6001 or C3. Abstracts are represented as agency ± SD from at atomic two absolute abstracts and bidding about to nontreated control; n = 4 (A) and n = 7 to 9 (B). Statistical differences were afflicted by Mann-Whitney U test. *P < 0.05; ***P < 0.001.
Loss of action of TREM2 is associated with NHD—a attenuate backward ataxia characterized by early-onset dementia with analytic presentation agnate to the behavioral accession of FTD (1). Recently, two absolute genome-wide affiliation studies affiliated missense variants in TREM2 to an added accident for developing late-onset AD (4, 5). Subsequent studies accustomed the affiliation in several AD cohorts (27, 28) and added continued the award to added neurodegenerative disorders including FTD (6, 8, 9), PD (6), and ALS (7). Although TREM2 variants are attenuate (population abundance ~0.3%), the aftereffect admeasurement (odds arrangement >3) is agnate to that for APOEε4; in silico predictions advance a apparent damaging aftereffect of the articular variants on TREM2 protein action (4, 8). Biochemical and corpuscle biological analyses of two FTD-associated TREM2 variants (p.Y38C and p.T66M) adumbrated misfolding of TREM2 followed by inhibition of corpuscle apparent carriage (Figs. 2 to 4). Bargain apparent acknowledgment of TREM2 leads to broken phagocytosis (Fig. 6), a award that is constant with a abbreviate ambit RNA–mediated TREM2 altercation phenotype (14). Admitting the FTD- and FTD-like–associated mutations, as able-bodied as the mutations of the conserved cysteine residues, displayed a actual astringent and constant biochemical and anatomic phenotype, the AD-associated p.R47H alteration showed a abundant weaker aftereffect on maturation, secretion, and phagocytic activity. Whether this is due to a weaker accustomed aftereffect of this TREM2 accession or rather a altered cellular apparatus of action charcoal to be investigated. Notably, the p.R47H alteration is advised to be a accident agency for AD (4, 5), admitting the added advised variants are adroit mutations back in the homozygous accompaniment (2). Furthermore, we empiric that the TREM2 p.R47H aberrant is bidding at lower rates, suggesting that this accession may be ambiguous and decumbent to degradation.
Our abstracts advance that bargain TREM2 action impairs phagocytosis, which may accord to neurodegeneration through altered mechanisms. First, bargain phagocytosis may anticipate engulfment of cellular debris, which could aftereffect in a abiding anarchic acknowledgment (12). Second, amyloidogenic seeds, which are anticipation to prime neurodegeneration (29), may additionally be alone in a TREM2-dependent manner. Third, in the case of AD, TREM2 is up-regulated about amyloid plaques (4, 30), and it is appetizing to brainstorm that this reflects a aegis apparatus to abolish exceptionable protein aggregates. In band with this hypothesis, phagocytosis assays appliance preaggregated Aβ1–42 as a ligand advance that TREM2 is absolutely able of removing amyloidogenic protein aggregates (Fig. 6, H to J). Finally, ER assimilation of aberrant TREM2 may additionally account ER stress, which could affect action and adaptation of microglia accurately in the case of the p.T66M and p.Y38C mutations.
TREM2 is a blazon I transmembrane glycoprotein that has been apparent to shuttle to and from the claret film in microglial beef aloft corpuscle dispatch by ionomycin or interferon-γ (31). Corpuscle apparent announcement of TREM2 can be adapted by either phagocytic receptor recycling (23) or ectodomain address (Figs. 1, 4, and 7) during which sTREM2 bits are appear from the cell. Whether sTREM2 has a paracrine signaling action or serves as a adversary for TREM2 ligands, as is the case for TREM1 (12), charcoal unclear. However, such functions would additionally be compromised by the bargain beard of TREM2 variants advised here. sTREM2 can be readily detected by ELISA-based methods (16) and could accordingly serve as a accessible brand for neurodegenerative disorders in the future, although added validation in added accommodating cohorts is needed. A accommodating with an FTD-like affection associated with the p.T66M alteration showed no sTREM2 in either CSF or plasma. Constant with a accident of action of TREM2, homozygous DAP12 deletions are additionally begin in patients with NHD (1, 32). If these after-effects are accustomed in a abundant beyond sample of patients, sTREM2 concentrations in CSF potentially could be acclimated to awning for TREM2 homozygous missense mutations in an akin way to progranulin alteration carrier screening (33–35). Furthermore, in a cross-sectional appraisal of sTREM2 concentrations in CSF of FTD patients, we begin decidedly bargain concentrations of sTREM2 in FTD patients compared to neurologically accustomed controls, added acknowledging a TREM2 loss-of-function apparatus in the pathogenesis of AD and FTD. Although the overlap amid groups precludes the accustomed appliance of sTREM2 in analytic settings, we accept that CSF sTREM2 deserves added appraisal as a abeyant brand for neurodegenerative diseases, apparently in aggregate with added yet to be articular markers. Finally, our allegation advance that TREM2-dependent phagocytosis could be modulated, appropriately aperture the aperture for new ameliorative strategies. Indeed, as a affidavit of principle, we showed that this can be accomplished by blocking ADAM proteases (Fig. 7). However, ameliorative appliance would crave a chase for compounds that selectively and accurately enhance TREM2 action afterwards interfering with the announcement of added proteins or capital signaling pathways.
The aboriginal allotment of this abstraction was advised to actuate whether TREM2 missense mutations, which afresh accept been articular as accident agency for several neurodegenerative diseases, adapt maturation and processing of TREM2 and ultimately account a accident of TREM2 protein function. We advised TREM2 processing and action appliance several corpuscle ambit including HEK293 Flp-In cells, microglial corpuscle ambit (BV2 cells), and primary microglial beef acquired from Trem2−/− mice. The added aim was to abstraction the sTREM2 concentrations in CSF and claret of patients diagnosed with FTD-like syndrome, FTD, or AD and analyze them to those of advantageous controls. For these purposes, we performed a cross-sectional abstraction in which we abstinent sTREM2 in CSF and claret samples from six specialized acoustic barometer centers. We advised all samples accessible from these centers; we did not accomplish a priori abacus of sample size. Analytic diagnoses were fabricated according to internationally accustomed criteria. In the final analysis, we afar those ascendancy individuals and FTD patients who had an AD CSF contour authentic by the Mattsson et al. blueprint (36), and, conversely, we afar those clinically diagnosed AD patients who did not accept an AD CSF profile. sTREM2 abstracts were performed in an ELISA-based appraisal that we developed. The abstracts were fabricated in a addled fashion. To abstraction the after-effects of TREM2 accident of function, we performed phagocytosis assays appliance three absolute targets (latex beads, E. coli bacteria, and Aβ1–42) and assuredly advised whether accentuation of TREM2 could adapt microglial function.
Flp-In 293 beef (HEK293 Flp-In; Life Technologies) were able in Dulbecco’s adapted Eagle’s average (DMEM) with Glutamax I, supplemented with 10% (v/v) fetal dogie serum (FCS), Zeocin (200 μg/ml), and penicillin/streptomycin (PAA Laboratories). Transfections of commutual DNA (cDNA) constructs were agitated out appliance Lipofectamine LTX with Plus Reagent according to the manufacturer’s recommendations. For abiding overexpression of animal TREM2 cDNA constructs, HEK293 Flp-In beef were cotransfected with the TREM2 cDNA constructs and pOG44 (Flp-recombinase announcement vector; Life Technologies) and called appliance hygromycin B (200 μg/ml). BV2 microglia beef (21) were able in DMEM with Glutamax I, supplemented with 10% (v/v) FCS and penicillin/streptomycin, and abiding TREM2-expressing corpuscle ambit were called appliance Zeocin (200 μg/ml). Corpuscle ambit were consistently monitored for Mycoplasma contagion via a PCR-based method, and all after-effects were abrogating throughout the advance of the study. If not declared otherwise, articles for corpuscle ability abstracts were acquired from Life Technologies.
Primary microglia were abandoned from postnatal day P5 to P6 abrasion accurateness appliance MACS Technology (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, academician cortices were dissected, freed from meninges, and dissociated by enzymatic assimilation appliance a Neural Tissue Dissociation Kit P. CD11b-positive microglia were magnetically labeled appliance CD11b MicroBeads, loaded assimilate a MACS column, and subjected to alluring separation. Abandoned microglia were argent assimilate 24-well bowl at a body of 8.5 × 104 beef per able-bodied and able in DMEM/F12 average (Life Technologies) supplemented with 10% heat-inactivated FCS (Sigma) and 1% penicillin/streptomycin and maintained in a humidified 5% CO2 incubator at 37°C. Afterwards 24 hours, able average was replaced with beginning average for 2 to 4 canicule until use for phagocytosis assays.
Bead uptake assays appliance HEK293 Flp-In beef durably cogent either wild-type or aberrant TREM2-DAP12 admixture constructs (25) were performed about as declared afore (23). Briefly, beef were argent in 24-well plates at a body of 5 × 104 beef and able overnight. Preopsonized [50% FCS in phosphate-buffered acrid (PBS)] acrylic chaplet (6 μm, internally absolute with the fluorophore Flash Red; Polysciences Inc.) were added to the beef at a absorption of 20 chaplet per corpuscle and incubated for 90 min at 37°C. As a abrogating control, phagocytosis was inhibited with 10 μM cytochalasin D 30 min afore accession of acrylic beads. Beef were harvested appliance TrypLE absorber (Life Technologies), done three times with fluorescence-activated corpuscle allocation (FACS) sample absorber (1% FCS and 0.02% sodium azide in PBS), and analyzed by breeze cytometry on a FACSCalibur breeze cytometer (BD Biosciences). Abstracts appraisal was performed appliance FlowJo adaptation 9.7.1.
Phagocytosis of fluorogenic E. coli particles (pHrodo Green, Molecular Probes) was analyzed appliance BV2 microglial cells, primary microglia or HEK293 Flp-In beef durably cogent either wild-type or aberrant TREM2-DAP12 admixture constructs (25). Briefly, beef were argent in 24-well plates at a body of 2 × 105 (HEK293 Flp-In), 1 × 105 (BV2), or 8.5 × 104 (primary microglia) beef and able for 24 to 48 hours. pHrodo E. coli bioparticles were attenuated in PBS to a absorption of 1 μg/μl, and a absolute of 50 μg of bioparticles was added per action and incubated for 60 min at 37°C. As a abrogating control, phagocytosis was inhibited with 10 μM cytochalasin D, which was added 30 min afore accession of pHrodo E. coli bioparticles. Beef were harvested by trypsinization, done two times with FACS sample buffer, and analyzed by breeze cytometry on a MACSQuant VYB breeze cytometer (Miltenyi Biotec). Abstracts appraisal was performed appliance the MACSQuantify software (Miltenyi Biotec).
Phagocytosis of aggregated FAM-labeled Aβ1–42 (Anaspec) was analyzed agnate to ahead declared methods (37). Briefly, FAM-labeled Aβ1–42 was aggregated brief at 37°C with agitation. Primary microglia, BV2, or HEK293 Flp-In beef were argent at 2 × 104, 1.5 × 104, 5 × 104 beef per well, respectively, in poly-l-lysine–coated black-walled 96-well plates (Greiner Bio One) and able overnight. Aβ was added to a final absorption of 0.5 μM (primary microglia) or 1.5 μM (HEK293 Flp-In and BV2) and incubated for 4 hours at 37°C. Extracellular Aβ1–42 was quenched with 100 μl of 0.2% trypan dejected in PBS (pH 4.4) for 1 min. Afterwards aspiration, fluorescence was abstinent at 485-nm excitation/538-nm discharge appliance a Fluoroskan Ascent FL bowl clairvoyant (Labsystems).
CSF samples were acquired by lumbar break afterward accepted procedures, calm in polypropylene tubes, and anon arctic at −80°C until use (36, 38). Six specialized acoustic barometer centers were complex in the study. AD was diagnosed according to the National Institute of Acoustic and Communicative Disorders and Stroke and the Alzheimer’s Ache and Related Disorders Affiliation belief (39). For patients recruited afore 2011, FTD was diagnosed according to the Neary accord belief (40). Thereafter, patients diagnosed of bvFTD (behavioral accession FTD) followed the new revised bvFTD belief (41), and those with semantic dementia or accelerating nonfluent aphasia accomplished the primary accelerating aphasia all-embracing accord belief (42). The ascendancy accumulation (n = 88) consisted of individuals accomplishing the afterward admittance criteria: (i) no acoustic or psychiatric antecedents, (ii) no amoebic ache involving the axial afraid arrangement afterwards all-encompassing analytic examination, and (iii) cerebral abasement was disqualified out afterwards appraisal by a neurologist with acquaintance in neurodegenerative dementias. Amid them, there were two patients who were diagnosed with disorders of the borderline afraid arrangement and two with depression. To advance the accurateness of the diagnosis, we afar those ascendancy individuals (6 of 94, 6.4%) and clinically diagnosed FTD patients (7 of 57, 12.3%) who had an AD CSF contour authentic by the Mattsson et al. blueprint (36) [(Aβ42/P-tau)/(3.694 0.0105 × T-tau)], and, conversely, we afar those clinically diagnosed AD patients who did not accept an AD CSF contour afterward the aloft blueprint (24 of 80, 30%). The abatement in sTREM2 concentrations in AD and FTD patients compared to ascendancy individuals was empiric both including and excluding those patients. Amid the FTD group, two patients agitated a C9orf72 echo amplification and three capacity agitated a progranulin (GRN) mutation. Again, the abatement in sTREM2 concentrations in FTD patients compared to ascendancy individuals was empiric both including and excluding the abiogenetic cases from the FTD group. All patients gave accounting abreast consent, and the abstraction was accustomed by the bounded belief committees of the accommodating centers.
To quantify the concentrations of sTREM2 in animal CSF, EDTA-plasma samples, or corpuscle ability supernatants, an ELISA for animal sTREM2 was accustomed appliance the Meso Scale Discovery SECTOR Imager 2400 analogously to ahead accustomed ELISAs (43). Streptavidin-coated 96-well plates were blocked brief at 4°C in blocking absorber [0.5% bovine serum albumin (BSA) and 0.05% Tween 20 in PBS (pH 7.4)]. For the apprehension of animal sTREM2, plates were incubated for 1 hour at allowance temperature with biotinylated polyclonal dupe anti-human TREM2 abduction antibiotic (0.25 μg/ml) (R&D Systems) adulterated in blocking buffer. Plates were done afterwards for four times with abrasion absorber (0.05% Tween 20 in PBS) and incubated for 2 hours at allowance temperature with samples adulterated 1:4 in appraisal absorber [0.25% BSA and 0.05% Tween 20 in PBS (pH 7.4)] supplemented with protease inhibitors (Sigma). A recombinant animal TREM2 protein (Hölzel Diagnostika) was adulterated in appraisal absorber in a bifold consecutive concoction and acclimated for the accepted ambit (concentration range, 4000 to 62.5 pg/ml). Plates were done three times for 5 min with abrasion absorber afore evolution for 1 hour at allowance temperature with abrasion monoclonal anti-TREM2 antibiotic (1 μg/ml) (Santa Cruz Biotechnology; B-3) adulterated in blocking buffer. Afterwards three added abrasion steps, plates were incubated with a SULFO-TAG–labeled anti-mouse accessory antibiotic (1:1000; Meso Scale Discovery) and incubated for 1 hour in the dark. Last, plates were done three times with ablution absorber followed by two abrasion accomplish in PBS and developed by abacus Meso Scale Discovery Read buffer. The ablaze discharge at 620 nm afterwards electrochemical dispatch was abstinent appliance the Meso Scale Discovery SECTOR Imager 2400 reader.
Spike recovery, linearity, interplate, and interday airheadedness for the animal sTREM2 ELISA was bent appliance both a committed CSF and claret sample (tables S2 and S3). Repeated freeze-thaw cycles had alone basal furnishings on sTREM2 concentrations in CSF (fig. S4C) and no aftereffect on sTREM2 concentrations in claret (fig. S4D). The specificity of the acclimated ELISA arrangement was added accurate by anti-TREM2 immunoblotting assuming aerial amount of alternation amid the ELISA readings and immunoreactivity on the immunoblot appliance an absolute anti-TREM2 antibiotic (fig. S4F).
To admeasurement murine sTREM2, the aforementioned action as absolute aloft was followed appliance biotinylated polyclonal sheep anti-mouse TREM2 (0.25 μg/ml) as abduction antibody, rat monoclonal anti-mouse TREM2 (1 μg/ml) as apprehension antibiotic (both R&D Systems), and a SULFO-TAG–labeled dupe anti-rat accessory antibiotic (1:1000; Meso Scale Discovery) as accessory antibody. As standard, a recombinant abrasion TREM2 protein (Hölzel Diagnostika) was used.
To quantify the levels of sTREM2 buried from HEK293 Flp-In or BV2 cells, conditioned media from biological replicates, calm as declared above, were analyzed in duplicates appliance either a bartering TREM2 ELISA according to the manufacturer’s recommendations (Sino Biological; Figs. 1B and 2B) or our anew accustomed animal (Fig. 4, A and B) or abrasion (Fig. 7, A and B) sTREM2 ELISA. The sTREM2 accepted curves were generated appliance the MasterPlex ReaderFit software (MiraiBio Group, Hitachi Solutions America) through a five-parameter logistic fit.
The χ2 appraisal was acclimated to analyze differences in absolute variables. One-way ANOVA followed by Tukey cavalcade hoc appraisal was acclimated to analyze commonly broadcast connected variables. Abstracts that did not chase a accustomed administration (including sTREM2 concentrations in CSF and plasma) were analyzed with nonparametric tests (Kruskal-Wallis followed by cavalcade hoc Mann-Whitney U test). To ascendancy for the aftereffect of abeyant confounders (gender, age, and centermost agent of the sample) on sTREM2 in CSF, we log-transformed this capricious to accomplish a accustomed distribution, and performed an ANCOVA. Statistical acceptation was set to 5% (α = 0.05). All tests were two-sided, and all abstracts were analyzed appliance the Statistical Package for the Social Sciences 20.0 (SPSS Inc.).
Materials and Methods
Fig. S1. Absence of TREM2 bits absolute C-terminal FLAG tag.
Fig. S2. Appraisal of maturation of TREM2.
Fig. S3. Characterization of BV2 beef durably overexpressing animal TREM2.
Fig. S4. Characterization of atypical sTREM2 ELISA.
Fig. S5. Broken phagocytosis by aberrant TREM2.
Table S1. Primers acclimated for RT-PCR analysis.
Table S2. Characterization of atypical sTREM2 ELISA.
Table S3. Spike accretion and breadth appraisal for CSF and claret sTREM2 ELISA.
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