Acta Neuropathologica (2021) 141:841–859 https://doi.org/10.1007/s00401-021-02296-1 ORIGINAL PAPER TREM2 expression in the brain and biological fluids in prion diseases Daniela Diaz‑Lucena1,2 · Niels Kruse3 · Katrin Thüne4 · Matthias Schmitz4,5 · Anna Villar‑Piqué1,2 · Jose Eriton Gomes da Cunha6 · Peter Hermann4 · Óscar López‑Pérez1,2 · Pol Andrés‑Benito1,2 · Anna Ladogana7 · Miguel Calero8,9 · Enric Vidal10 · Joachim Riggert11 · Hailey Pineau12 · Valerie Sim12 · Henrik Zetterberg13,14,15,16 · Kaj Blennow13,14 · Jose Antonio del Río17,18,19,20 · Alba Marín‑Moreno21 · Juan Carlos Espinosa21 · Juan María Torres21 · Raquel Sánchez‑Valle22 · Brit Mollenhauer23,24 · Isidre Ferrer1,2,25 · Inga Zerr4,5 · Franc Llorens1,2,4 Received: 23 February 2021 / Revised: 15 March 2021 / Accepted: 15 March 2021 / Published online: 21 April 2021 © The Author(s) 2021 Abstract Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune cell surface receptor that regulates microglial function and is involved in the pathophysiology of several neurodegenerative diseases. Its soluble form (sTREM2) results from shedding of the TREM2 ectodomain. The role of TREM2 in prion diseases, a group of rapidly progressive dementias remains to be elucidated. In the present study, we analysed the expression of TREM2 and its main sheddase ADAM10 in the brain of sporadic Creutzfeldt-Jakob disease (sCJD) patients and evaluated the role of CSF and plasma sTREM2 as a poten- tial diagnostic marker of prion disease. Our data indicate that, compared to controls, TREM2 is increased in sCJD patient brains at the mRNA and protein levels in a regional and subtype dependent fashion, and expressed in a subpopulation of microglia. In contrast, ADAM10 is increased at the protein, but not the mRNA level, with a restricted neuronal expression. Elevated CSF sTREM2 is found in sCJD, genetic CJD with mutations E200K and V210I in the prion protein gene (PRNP), and iatrogenic CJD, as compared to healthy controls (HC) (AUC = 0.78–0.90) and neurological controls (AUC = 0.73–0.85), while CSF sTREM2 is unchanged in fatal familial insomnia. sTREM2 in the CSF of cases with Alzheimer’s disease, and multiple sclerosis was not significantly altered in our series. CSF sTREM2 concentrations in sCJD are PRNP codon 129 and subtype-related, correlate with CSF 14-3-3 positivity, total-tau and YKL-40, and increase with disease progression. In plasma, sTREM2 is increased in sCJD compared with HC (AUC = 0.80), displaying positive correlations with plasma total-tau, neurofilament light, and YKL-40. We conclude that comparative study of TREM2 in brain and biological fluids of prion diseases reveals TREM2 to be altered in human prion diseases with a potential value in target engagement, patient stratification, and disease monitoring. Keywords TREM2 · Prion diseases · Creutzfeldt-Jakob disease · Microglia · Cerebrospinal fluid · Plasma Introduction ­(PrPc), ­PrPSc, which nucleates and self-propagates in the central nervous system (CNS) [3]. However, the mecha- Prion diseases (or transmissible spongiform encephalopa- nisms by which ­PrPSc causes neurodegeneration are not thies) are a rare group of invariably fatal neurodegenerative well-understood. dementias characterized by a rapid progression affecting Human prion diseases can be classified according to their humans and animals. The causative agent of prion dis- aetiology as sporadic Creutzfeldt-Jakob disease (sCJD); eases is the pathological form of the cellular prion protein genetic Creutzfeldt-Jakob disease (gCJD), Gerstmann-Sträu- ssler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI); and as iatrogenic Creutzfeldt-Jakob disease (iCJD), * Isidre Ferrer variant Creutzfeldt-Jakob disease (vCJD), and Kuru. sCJD 80982ifa@gmail.com is the most prevalent human prion disease and its typical * Inga Zerr clinical phenotype includes rapidly progressive dementia, ingazerr@med.uni-goettingen.de behavioural abnormalities, ataxia, extrapyramidal features, Extended author information available on the last page of the article 13 Vol.:(0123456789) 842 Acta Neuropathologica (2021) 141:841–859 and myoclonus. Eventually, sCJD patients fall into a state of expressed predominantly in cells of myeloid lineage, in line akinetic mutism before death [18, 54]. Neuropathologically, with its role in brain innate immune function [15, 28, 61]. sCJD is characterized by neuronal loss, spongiform change, No effect of genetic variation in TREM2 in the risk of CJD reactive astrocytosis, and microgliosis. These changes are has been identified [63]. Yet pioneering studies have shown accompanied by the accumulation of a protease-resistant increased microglial TREM2 mRNA expression in scrapie- form of host-derived prion protein ­(PrPres) [17]. infected mice and prion-inoculated TREM2 knockout mice, A clinical diagnosis of probable sCJD is achieved by the with attenuated microglia response compared to controls combination of clinical symptoms and signs, neuroimaging, but no alterations in prion pathogenesis in terms of disease and cerebrospinal fluid tests (CSF) [23, 26]. The most rel- onset, disease duration, and presence of pathological PrP evant CSF biomarkers are surrogate neuronal damage mark- [80]. TREM2 expression in brain and biological fluids of ers 14-3-3, total-tau (t-tau), and α-synuclein (a-syn), while human prion diseases and its potential role as a candidate the real-time quaking induced conversion (RT-QuIC) assay biomarker of prion pathogenesis have not been explored. is a prion-specific assay allowing identification of the patho- logical prion protein in the CSF [70]. Recently, some blood biomarkers have been shown to be suitable in discriminating Material and methods sCJD cases but their accuracy in the differential diagnosis of sCJD has not been determined [30, 66, 72]. Therefore, they Antibodies and reagents are not yet included in clinical practice. Importantly, the use of surrogate CSF biomarkers is not limited to diagnostic pur- Human TREM2 goat polyclonal antibody (RD Systems, poses; rather, a potential use as prognostic, predictive, moni- AF1828) was used diluted 1:20 for immunohistochemistry toring, and response biomarkers has been proposed [35, 46, and at a dilution of 1:200 for western blotting. The anti- 65, 71]. Therefore, the characterization of the best putative body anti-TREM2 Abcam [EPR20243] ab209814 diluted application for each biomarker as well as the identification 1:100 was used for immunohistochemistry for comparative of biomarkers targeting the complete pathological signatures purposes. Human ADAM10 antibody (Abcam, ab1997) was of prion disease will greatly enhance the rational develop- used at a dilution of 1:75 for immunohistochemistry and ment and interpretation of future therapeutic interventions. 1:500 for western blotting. Monoclonal anti-β-actin antibody In this regard, the observation that exacerbated inflamma- (Sigma-Aldrich, A5316) was used at 1:30,000 as a protein- tory profiles is an early pathogenic event in prion diseases loading control in western blotting. For double-labelling [2, 34] has led to the search for surrogate biomarkers of immunofluorescence and confocal microscopy visualization, neuroinflammation for early diagnosis. In this context, glial- TREM2 was combined with the microglial marker IBA1 specific proteins such as the S100 calcium-binding protein diluted 1:1000 (Wako, 019-19,741). B (S100B), chitinase-3-like protein 1 (CHI3L1/YKL-40), chitotriosidase 1 (CHIT1), and the glial fibrillary acidic pro- Brain samples tein (GFAP) have been reported to be increased in the CSF of sCJD [2, 25, 37, 58]. Brain tissue was obtained from the Institute of Neuropa- In recent years, strong evidence of a link between TREM2 thology HUB-ICO-IDIBELL Biobank and the Hospital function in the innate immune system and the pathogen- Clinic de Barcelona-IDIBAPS Neurological Tissue Bank esis of neurodegenerative diseases has been documented. following the Spanish legislation guidelines on this mat- Although rare in the population, certain TREM2 variants ter (Real Decreto de Biobancos 1716/2011) and approval are associated with increased risk of developing neurode- of the local ethics committees. Processing of brain tissue generative diseases such as Alzheimer’s disease (AD) [21, has been detailed elsewhere [16]. The post-mortem interval 26], frontotemporal dementia [20, 56], Parkinson’s disease between death and tissue processing was between 1 h 30 min [56], and amyotrophic lateral sclerosis [7], presumably as and 24 h. One hemisphere was immediately cut in coronal a consequence of TREM2 loss of function [12]. In this sections, 1-cm thick, and selected areas of the encephalon regard, TREM2 mutations led to reduced phagocytic func- were rapidly dissected, frozen on metal plates over dry ice, tion [64], while injection of sTREM2 in an AD mouse model placed in individual air-tight plastic bags, numbered with enhanced microglial proliferation, migration, clustering in water-resistant ink, and stored at − 80 °C until use for bio- the vicinity of amyloid plaques, and the uptake and degrada- chemical studies. The other hemisphere was fixed by immer- tion of β-amyloid [79]. In AD patients, increased TREM2 sion in 4% buffered formalin for 3 weeks for morphologic levels have been reported in the CSF [22, 24, 41, 42, 55], study. Neuropathological examination was performed in together with positive immunoreactivity in the microglia every case on formalin-fixed, paraffin-embedded samples. associated with β-amyloid plaques and in neuritic pathology- De-waxed paraffin Sects. 4 microns thick were stained with enriched areas [41]. It is currently accepted that TREM2 is haematoxylin and eosin, periodic acid Schiff, Congo red, 13 Acta Neuropathologica (2021) 141:841–859 843 and Klüver-Barrera, or processed for immunohistochemis- composed of controls (n = 15 FC, n = 12 CB), sCJD (n = 11 try using anti-β-amyloid, p-tau (clone AT8), α-synuclein, FC, n = 12 CB), and sCJD VV2 (n = 11 FC, n = 10 CB). The αB-crystallin, TDP-43, P-TDP-43, ubiquitin, p62, glial sFTLD group consisted of samples from the FC of con- fibrillary acidic protein, CD68, and IBA1, as previously trols (n = 17) and sFTLD-TDP (n = 16). Details of the brain reported [16]. Neuropathological diagnosis of Alzheimer’s samples used for q-PCR are summarized in Supplementary disease (AD) was categorized following Braak stages of neu- Table 1. Samples for western blotting were obtained from 10 rofibrillary tangle pathology adapted to paraffin sections [5, controls, 8 sCJD MM1, and 8 sCJD VV2 cases. 6]. Cases with Frontotemporal lobar degeneration with TDP- Triggering receptor expressed on myeloid cells 2 immu- 43 pathology (FTLD-TDP) were diagnosed following estab- nohistochemistry was performed on de-waxed paraffin lished neuropathological criteria [43]. Post-mortem neuro- sections of controls (n = 10; 7 M, 3 F; mean age ± SD: pathological examination confirmed the diagnosis of sCJD 57.9 ± 12.5), CJD MM1 (n = 7; 6 M, 1 not available; mean cases according to established criteria [49, 50]. The analysis age ± SD: 63.8 ± 13.9), AD V-VI (n = 3; 3 F; mean age ± SD: of the codon129 genotype of PrP gene (PRNP) (methionine 77.3 ± 4.1), sFTLD-TDP (n = 3; 3 M; mean age ± SD: or valine) was performed after isolation of genomic DNA 72 ± 7.1), DLB (n = 3; 2F,1 M; mean age ± SD: 78.3 ± 4.9), from blood samples according to standard methods. sCJD ALS (n = 4; 3 F, 1 M; mean age ± SD: 62 ± 12.5), white type 1 or type 2 classification was performed with western matter with plaques of relapsing remitting cases of multiple blotting. Cases with associated pathologies such as vascu- sclerosis (MS, n = 1 M, 52 years), and vascular dementia lar diseases, infections of the nervous system, neoplastic with subacute areas of infarction (DM, n = 3 M; 72) (see diseases affecting the nervous system, systemic and central Supplementary table 3). immune diseases, metabolic syndrome, hypoxia, and pro- longed axonal states such as those occurring in intensive Cerebrospinal fluid and plasma samples care units were excluded from the present study. Since a percentage of CJD cases have concomitant NFT Cerebrospinal fluid (CSF) samples (Table 1) from healthy pathology at stages 0-III of Braak, with occasional amy- control individuals (HC, n = 48), patients with non-primarily loid plaques (stage A of Braak), control cases in the present neurodegenerative neurological diseases (ND, n = 64), AD series are not controls without NFT pathology but rather (n = 35), and sCJD (n = 139) were collected at the Clinical age-matched controls with NFT pathology at stages 0-III Dementia Centre and the National Reference Centre for CJD without neurological symptoms. Mild small blood vessel Surveillance at the University Medical Centre of Göttingen disease and occasional status cribosus occurred in the ‘con- (Germany) and at the Alzheimer’s Disease and Other Cogni- trol’ and ‘pathological’ groups. tive Disorders Unit, Hospital Clínic de Barcelona, Barcelona Molecular studies were carried out in frontal cortex (Spain). Patients with sCJD, classified as probable (n = 47) area 8 (FC) and the cerebellum (CB). The CJD group was or definite (n = 92) cases according to diagnostic consensus Table 1  Biomarker characteristics of the cases used in the CSF cohort of study. Diagnostic groups included controls (HC, ND), prion diseases (sCJD, gCJD-E200K, gCJD-V210I, FFI and iCJD), and AD cases n Sex (f/m) Age sTREM2 (pg/ 95% CI tau (pg/mL) 14-3-3 p value mL) Mean + SD Mean + SD Mean + SD Pos/neg sTREM2 vs. sTREM2 vs. age sex HC 48 26/22 64 ± 15 1898 ± 842 1653–2143 246 ± 132 0/48 0.26 0.20 ND 64 39/25 66 ± 9 2321 ± 1125 2040–2602 441 ± 410 13/49 0.87 0.61 sCJD 139 73/66 66 ± 11 6975 ± 8523 5544–8403 7337 ± 7658* 118/21 0.83 0.26 gCJD—E200K 57 35/22 60 ± 10 6341 ± 8128 4184–8498 6548 ± 5688 49/8 0.13 0.91 gCJD—V210I 34 18/16 66 ± 10 8231 ± 10,257 4652–11,810 10,548 ± 9985 27/7 0.18 0.63 FFI—D178N- 26 9/17 50 ± 12 2871 ± 2704 1779–3963 993 ± 1715 4/22 0.22 0.19 M iCJD 10 5/5 50 ± 11 4709 ± 2712 3025–6036 12,171 ± 10,053 8/2 0.42 0.44 AD 35 22/13 67 ± 10 2896 ± 1603 2345–3447 633 ± 401 NA 0.41 0.25 Number of cases (n), age [mean value ± standard deviation (SD)], sex distribution (f female, m male), CSF sTREM2 [mean value ± SD, and 95% confidence interval (95% CI)] and t-tau (mean value ± SD) concentrations as well as 14-3-3 positivity (pos/positive, neg/negative) are indicated. Inconclusive 14-3-3 tests were considered as negative. Significant associations (p value) between sTREM2 with sex and age for each diagnostic group are reported. *For 13 cases, t-tau data were not available. (NA not available) 13 844 Acta Neuropathologica (2021) 141:841–859 criteria [76], genetic CJD (n = 91), iatrogenic CJD (n = 10), cases, n = 86 cases) of the University Medical Centre Göt- and fatal familial insomnia (FFI, n = 26) were collected in tingen (Germany) under standardized pre-analytical con- the following CJD reference centres: (1) Clinical Dementia ditions. The healthy control (HC) group was composed of Centre and the National Reference Centre for CJD Surveil- healthy blood donors without any relevant clinical findings lance at the University Medical Centre, Göttingen, Germany, (Table 2). (2) Alzheimer’s Disease and Other Cognitive Disorders For the analysis of CSF sTREM2 concentrations at differ- Unit, Hospital Clínic, Barcelona, Spain, (3) National Cen- ent disease stages in sCJD, we divided the time from lum- tre of Microbiology-Carlos III Institute of Health, Madrid, bar puncture (LP) or blood uptake to disease onset in each Spain, and (4) Istituto Superiore di Sanità, Rome, Italy. The patient by the total duration of the disease. Then, samples diagnosis of genetic prion diseases was carried out accord- were grouped into three categories according to whether ing to surveillance criteria after prion protein gene (PRNP) they underwent LP/blood uptake in the first (time of LP to analysis [31] and following World Health Organization disease onset/total duration of the disease < 0.33), second (WHO) criteria [73]. Iatrogenic CJD was diagnosed accord- (0.33–0.66), or third (> 0.66) stage of the disease, as previ- ing to established WHO criteria [73]. Nine iatrogenic cases ously reported [59]. were associated with dura mater grafts and one with corneal transplantation. AD cases were diagnosed according to the CSF and plasma tests National Institute on Aging—Alzheimer’s Association work- groups (NIA-AA) criteria [45]. Soluble TREM2 (sTREM2) concentrations in the CSF and In addition, CSF was obtained from patients with relaps- plasma were quantified with a previously described immu- ing/remitting multiple sclerosis [69] at the Multiple Sclero- noassay [29] using the electrochemiluminescence-based sis Unit, Service of Neurology, Bellvitge University Hospital MesoScale Discovery platform. Inter- and intra-assay (n = 16), and the Clinical Dementia Centre and the National coefficients of variation in our study were below 12% and Reference Centre for CJD Surveillance at the University 10%, respectively. CSF from MS cases was assessed in two Medical Centre of Göttingen (Germany) (n = 13 (Supple- series. In the first, the method was the previously described mentary table 4). immunoassay [29], while for the second series (Bellvitge) Cerebrospinal fluid tests in healthy controls (HC) was assessment was made using the Human TREM2 ELISA Kit obtained from patients for knee surgical procedures under (Abcam, ab224881). CSF total-tau (t-tau) was quantified spinal anaesthesia. using a commercially available colorimetric enzyme-linked The ND group was composed of cases diagnosed with immunosorbent assay (INNOTEST hTAU-Ag from Fujire- neurological diseases not associated with neurodegenera- bio). CSF was analysed for the presence of 14 to 3-3 protein tive pathology and included the following diagnostic groups: using western blotting [75]. CSF and plasma YKL-40 were psychosis, paranoid psychosis, bipolar disorder, schizophre- quantified using the MicroVue YKL-40 ELISA kit from nia, ischemic stroke, multiple infarct, epilepsy, meningitis, Quidel. Plasma neurofilament light (NfL) and t-tau concen- alcohol abuse, vertigo, acute or chronic headache, pain syn- trations were measured using commercially available assays dromes, acute hypoxia, polyneuropathy, cerebral lymphoma, on the Single molecule array (SIMOA) HD-1 instrument astrocytoma, and paraneoplasic syndromes. ND cases were (Quanterix). The analysts were masked to clinical data. diagnosed according to acknowledged standard neurologic clinical and para-clinical findings based on the International Animal experimentation Classification of Diseases 10th Revision definitions. Blood was collected in Plasma-EDTA tubes at the We conducted animal experiments in accordance with the Department of Transfusion Medicine (healthy controls, HC; Code for Methods and Welfare Considerations in Behav- n = 97 cases) and at the Department of Neurology (sCJD ioural Research with Animals (Directive 2010/63/EU) Table 2  Demographic and n Sex (f/m) Age sTREM2 pg/mL) 95% CI p values biomarker characteristics of the cases used in the plasma cohort Mean + SD Mean + SD sTrem2 vs. Sex sTrem2 vs. age study HC 97 23/74 64 ± 5 4409 ± 3961 3611–5208 0.58 0.12 sCJD 86 52/34 65 ± 8 8981 ± 8932 7066–10,896 0.94 0.11 Diagnostic groups included HC and sCJD. Number of cases (n), age: mean value ± standard deviation (SD), sex distribution (f female, m male), and plasma sTREM2 concentrations: mean value ± SD and 95% confidence interval (95% CI) are indicated. Significant associations (p value) between sTREM2 with sex and age for each diagnostic group are indicated 13 Acta Neuropathologica (2021) 141:841–859 845 and made every effort to minimize suffering. Experiments of the RNA samples was carried out using the High-Capac- developed in the Centro de Investigación en Sanidad Ani- ity cDNA Reverse Transcription kit (Applied Biosystems) mal–Instituto Nacional de Investigación y Tecnología following the supplier’s instructions. Quantitative RT-PCR Agraria y Alimentaria (Madrid, Spain) were evaluated by assays were performed in duplicate on cDNA samples in the Committee on the Ethics of Animal Experiments of the 384-well optical plates with ABI Prism 7900 Sequence Instituto Nacional de Investigación y Tecnología Agraria Detection System (Applied Biosystems, Life Technologies). y Alimentaria and approved by the General Directorate of The reactions were carried out using 20xTaqMan Gene the Madrid Community Government (permit no. PROEX Expression Assays and 2xTaqMan Universal PCR Mas- 094/18). The tg340 and tg361 mouse lines expressing about ter Mix (Applied Biosystems) with the following probes: four-fold level of human PrPM129 and PrPV129 on a mouse CD68 Hs00154355_m1, TREM2 human Hs00219132_m1, PrP null background were used as mouse models of sCJD TREM2 mouse Mm04209424_g1, and ADAM10 human MM1 and sCJD VV2 subtypes. Individually identified 6-to- Hs00153853_m1. The reactions were conducted using the 10-week-old mice were anaesthetized and inoculated with following parameters: 50 °C for 2 min, 95 °C for 10 min, 2 mg of a 10% (w/v) brain homogenate of sCJD MM1 (in and 40 cycles with 95 °C for 15 s and 60 °C for 1 min. The the tg340 mouse line) and sCJD VV2 (in the tg361) in the fold change was determined by the ΔΔCt method, in which right parietal lobe using a 25-gauge disposable hypodermic each target gene is normalized to its endogenous control needle. Control animals for each genotype were inoculated to obtain the ΔCt. Afterwards, each ΔΔCt is determined with control brain homogenates. Mice were observed daily by subtracting the mean-ΔCt of the control group samples and their neurological status was assessed weekly. At 80, to the ΔCt of each sample. Finally, these ΔΔCt values are 120, or 180 days post-inoculation (dpi) animals were eutha- used as the negative exponent with base 2 to obtain the fold nized, necropsy was performed, and the brain was removed. change per sample. Mean fold change values were analysed Four to eight animals per time point and condition were used with appropriate statistical test indicated in each figure for RT-qPCR assays. using GraphPad Prism 6.01. Gene expression levels were normalized to the housekeeping genes Glucuronidase beta Western blotting (GUSB) Hs00939627_m1 NM_000181.3 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs03929097_g1 Human tissues were lysed in RIPA lysis buffer RIPA (NaCl NM_001289745.1 for human and, GAPDH Mm99999915_ 1 M, Nonidet P-40 1%, sodium deoxycholate 0.5%,SDS g1 NM_001289726.1 for mouse (Applied Biosystems). 0.1%, Tris 50 mM, pH 7.4) supplemented with protease, and phosphatase inhibitors (Roche, GE). After centrifugation at Immunohistochemistry 14,000g for 20 min at 4 °C, supernatants were quantified for protein concentration (BCA, Pierce), mixed with SDS- De-waxed sections, 4 microns thick, were processed for PAGE sample buffer, boiled, and subjected to 4–12% BisTris immunohistochemistry. The sections were boiled in citrate Gels ­(NuPAGE™ 4 to 12%, Bis–Tris, 1.5 mm, Mini Protein buffer to retrieve antigenicity, followed by endogenous per- Gel, 15-well, ThermoFisher NP0336BOX). Gels were trans- oxidase blockage (Dako Real peroxidase blocking solution ferred onto PVDF membranes and processed for specific S2023). All sections were incubated overnight at 4 °C with immunodetection. Membranes were incubated overnight at primary antibodies diluted in Dako Real Antibody Diluent 4 °C with primary antibodies and with appropriate horserad- (Dako S2022). ish peroxidase-conjugated secondary antibody (anti-mouse/ Primary antibody anti-human TREM2 (RD Systems anti-rabbit HRP-IgG) diluted at 1:2000 (Dako) for 1 h at AF1828) diluted 1:20, anti-human TREM2 (Abcam, room temperature. Chemiluminescence immunodetection ab209814) diluted 1:100, and anti-human ADAM10 was performed using the ECL-Amersham (GE Healthcare) (Abcam, 1997) diluted 1:150 were used. Detection was kit. Densitometry was carried out with the ImageJ software achieved by incubation with R.T.U. Biotinylated Universal and values were normalized using β-actin levels. Antibody (Abcam ab64257) for 10 min at room temperature, followed by R.T.U. HRP-Streptavidin (Abcam ab64269). Real‑time polymerase chain reaction (qPCR) The peroxidase reaction was visualized with diaminoben- zidine and ­H2O2. Control of the immunostaining included RNA from different human and mouse brain regions was omission of the primary antibody. purified using RNeasy Plus Mini Kit (Qiagen, 74,136), which eliminates genomic DNA contamination. RNA integ- Immunofluorescence rity was assessed with the RNA Integrity Number (RIN value) determined with the Agilent 2100 Bioanalyzer (Agi- De-waxed sections, 4 microns thick, were boiled in citrate lent) (RIN values between 4 and 9.2). Reverse transcription buffer to enhance antigenicity, and afterwards were stained 13 846 Acta Neuropathologica (2021) 141:841–859 with a saturated solution of Sudan black B (Merck, DE) in PBS, and incubated at 4ºC overnight with combina- to block auto-fluorescence of lipofuscin granules present tions of primary antibodies. After washing, the sections in cell bodies. Then they were rinsed in 70% ethanol and were incubated with Alexa-488 and Alexa-555 (Molecu- washed in distilled water. All sections were blocked at lar Probes, Eugene, Oregon, USA) fluorescence secondary room temperature with 10% foetal bovine serum diluted ­ RAQ5™ (dilution antibodies. Nuclei were stained with D 1:2,000, BioStatus, Loughborough, UK). After washing, 13 Acta Neuropathologica (2021) 141:841–859 847 ◂Fig. 1  TREM2 expression in sCJD brain tissue. RT-qPCR analysis of Results TREM2 in: a the frontal cortex (FC) and b cerebellum (CB) of con- trol, sCJD MM1, and sCJD VV2 cases. GAPDH was used for normal- ization. FC (controls n = 15, sCJD MM1; n = 11, sCJD VV2; n = 11), TREM2 and ADAM10 expression in the brain of sCJD CB (controls n = 12, sCJD MM1; n = 12, sCJD VV2; n = 10). c RT- patients qPCR analysis of TREM2 in the FC of sCJD (MM1 and VV2), and FTD-TDP. For each neurodegenerative dementia, data were compared Expression of TREM2 was analysed in the frontal cortex to age and sex-matched controls. CJD (controls n = 14, sCJD (MM1 and VV2); n = 22), FTD-TDP-43 (controls n = 17, FTD-TDP-43; (FC) and cerebellum (CB) of sCJD MM1 and VV2 cases. n = 16). d Representative western blot analysis of TREM2 in the FC With RT-qPCR analysis, TREM2 mRNA was significantly and CB of control, sCJD MM1, and sCJD VV2 cases. TREM2 imma- increased in the FC of sCJD MM1 (n = 14, p < 0.001, fold ture (TREM2i) and mature (TREM2m) forms are indicated. For nor- change 6.1) and VV2 (n = 14, p < 0.001, fold change 3.5) malization β-actin was used. Graphical representation of western blot data acquired from the analysis of FC (controls n = 8, sCJD MM1; cases compared to controls (n = 15). TREM2 mRNA expres- n = 8, sCJD VV2; n = 10) and CB (controls; n = 8, sCJD MM1; n = 8, sion was higher in sCJD cases possessing the PRNP gene sCJD VV2; n = 8) is shown; *p < 0.05, **p < 0.01, ***p < 0.001. e codon 129 MM1 as compared to VV2 genotype (p < 0.05) TREM-2 immunohistochemistry in frontal cortex and cerebellum (Fig. 1a). In the CB, TREM2 mRNA was significantly in control cases, and then in frontal cortex in CJD MM1. In control and CJD cases, TREM2 immunoreactivity is localized in the blood increased in sCJD VV2 (n = 10, p < 0.001) and MM1 (n = 12, vessels (thick arrows); in CJD, TREM2 immunoreactivity is also p < 0.05) compared to controls (n = 12) (Fig. 1b). TREM2 observed in glial cells with the morphology of microglia (long thin levels were normalized to GAPDH, and similar results arrows); scale bar = 30 µm. f TREM2 double-labelling immunofluo- were obtained for GUSB normalization (data not shown). rescence and confocal microscopy in FC of sCJD showing TREM2 localization in a subset of microglial cells identified with the anti- Increased TREM2 mRNA expression in the FC in sCJD was body Iba1 (white arrows). Nuclei are stained with ­DRAQ5™ (blue); significant (n = 28, p < 0.001) when compared with FTLD- scale bar = 20 µm. g Correlation analysis between TREM2 and CD68 TDP (n = 16) (Fig. 1c). mRNA in the FC of sCJD cases (n = 28). cc: correlation coefficient Western blot analysis revealed significantly increased pro- tein levels of mature and total TREM2 (mature plus imma- the sections were mounted in Immuno-Fluore mounting ture forms) in the FC of sCJD cases compared to controls medium (ICN Biomedicals, Irvine, CA, USA), sealed, (p < 0.001, control vs. sCJD MM1 and p < 0.01, control vs. and dried overnight. Sections were examined with a Leica sCJD VV2 for total TREM2) (Fig. 1d). The expression of TCS-SL confocal microscopy. mature and total TREM2 was significantly higher in sCJD MM1 cases compared to sCJD VV2 (p < 0.05). In the CB, Statistical analysis mature and total TREM2 was significantly higher in sCJD cases compared to controls (p < 0.05 in all comparisons) For brain tissue data, in two group comparisons, non-para- (Fig. 1d). In both brain regions, 10 controls, 8 sCJD MM1, metric Mann–Whitney-U tests were used. For comparisons and 8 sCJD VV2 cases were used. between multiple groups, Kruskal–Wallis tests followed by In immunohistochemistry study of control brains using Dunn’s post-hoc test or one-way ANOVA followed by Tukey the TREM2 polyclonal RD Systems, AF1828H antibody dis- post-hoc test were applied after testing for normality. Bio- closed immunostaining in the blood vessel walls, with no marker data were log-transformed, and linear models were immunostaining of neurons or glial cells (data not shown). built controlling for the effect of age and sex. To assess the Using the same antibody, TREM2 immunoreactivity in diagnostic accuracy of sTREM2, receiver operating char- sCJD MM1 brains was observed in the blood vessel walls acteristic (ROC) curve analyses were carried out and areas and in microglia; neurons were not immunostained with under the curve (AUC) with confidence intervals (95% CI) this antibody (Fig. 1e). De-waxed paraffin sections of AD, were calculated. Spearman rank correlation coefficients were DLB, MS, sALS, and VD cases showed TREM2 immuno- used to assess associations between continuous biomarker reactivity in the blood vessels and microglia surrounding levels. Statistical comparison between ROC curves was β-amyloid plaques in AD, lipid-laden macrophages in suba- made with the pROC R package [57]. Longitudinal analysis cute MS plaques, macrophages and a few mononuclear cells of sTREM2 along disease stages was conducted with nlme in subacute infarcts, and a few microglia in the spinal cord R package [53] using a multilevel linear model that assumed in sALS. Neurons were not stained with this antibody (data a fixed slope for disease stage. not shown). To validate immunohistochemical data, double- labelling immunofluorescence against TREM2 and Iba-1 in the FC of sCJD MM1 cases disclosed TREM2 localization in a subpopulation of microglial cells as revealed with the IBA1 antibody (Fig. 1f). Additionally, TREM2 mRNA in 13 848 Acta Neuropathologica (2021) 141:841–859 FC of sCJD cases correlated positively with the macrophage but the TREM2 Abcam antibody is a discontinued product marker CD68 (n = 28, cc = 0.4701, p = 0.0116) (Fig. 1g). and no longer available. In contrast, immunohistochemistry with the anti-TREM2 Next, we explored whether ADAM10, the main TREM2 Abcam antibody showed neuronal immunoreactivity in con- sheddase responsible for its regulated intramembrane pro- trol and diseased cases, in addition to immunostaining of the teolysis [60], was also altered in sCJD patients. With RT- blood vessel walls and rare microglia (data not shown). We qPCR analysis, no alterations in ADAM10 mRNA levels tried to reproduce these observations using another batch were observed in sCJD, either in the FC (controls, n = 15; Fig. 2  ADAM10 expression in sCJD brain tissue. a RT-qPCR analysis of ADAM10 in the fron- tal cortex (FC) and cerebellum (CB) of control, sCJD MM1, and sCJD VV2 cases. GAPDH was used for normalization. FC (controls n = 15, sCJD MM1; n = 11, sCJD VV2 n = 10), CB (controls n = 12, sCJD MM1; n = 12, sCJD VV2 n = 10). b Representative western blot analysis of ADAM10 in the FC and CB of control, sCJD MM1, and sCJD VV2 cases. ADAM10 precursor (ADAM10p) and mature (ADAM10m) forms are indicated. β-actin was used for normalization. Graphical representation of western blot data acquired from the analysis of FC (controls n = 12, sCJD MM1; n = 12, sCJD VV2; n = 12) and CB (controls; n = 9, sCJD MM1; n = 10, sCJD VV2; n = 10); *p < 0.05, **p < 0.01, ***p < 0.001. c ADAM10 immunohistochemistry in the frontal cortex (FC), cerebellum (Cb), and subcortical white mat- ter (WM) in control and sCJD MM1. ADAM10 immunoreac- tivity is observed in neurons in the FC and granule cells (gr) of cerebellum, but Purkinje cells (P) are very weakly stained or not at all. No positive cells are distinguished in the white mat- ter; scale bar = 20 µm 13 Acta Neuropathologica (2021) 141:841–859 849 sCJD MM1, n = 14 and sCJD VV2, n = 10) or in the CB sex (Table 1), both when all cases were analysed together (controls, n = 12; sCJD MM1, n = 12 and sCJD VV2, n = 10) and when stratified between non-prion and prion diseases. (Fig. 2a). TREM2 levels were normalized to GAPDH, and Compared to HC and ND, sTREM2 concentrations were similar results were obtained for GUSB normalization (data significantly higher in all types of prion diseases, with the not shown). In contrast, ADAM10 precursor and mature exception of FFI cases (Fig. 4a). sTREM2 mean concentra- forms were increased in western blot in the FC of sCJD tions were higher in ND (2413 ± 1262 pg/mL) than in HC MM1 (p < 0.05 precursor, p < 0.001 mature) and sCJD VV2 (1901 ± 878 pg/mL). Accordingly, the AUC derived from the (p < 0.01 precursor, p < 0.001 mature) compared to con- sCJD vs. HC comparison (0.90) was higher than that for the trols (Fig. 2b). In the CB, ADAM10 levels were not signifi- sCJD vs. ND comparison (0.85) (Fig. 4b). An optimal cut- cantly different for sCJD compared to controls (p > 0.05 in off of 2650 pg/mL sTREM2 discriminated HC from sCJD all comparisons) (Fig. 2b). In both brain regions, 10 con- cases with 79% sensitivity and 94% specificity, while a cut- trols, 8 sCJD MM1, and 8 sCJD VV2 cases were used. The off of 2850 pg/mL sTREM2 discriminated ND from sCJD ADAM10 antibody showed suboptimal results for immu- cases with 77% sensitivity and 80% specificity. AUCs for the nohistochemistry; ADAM10 immunostaining was weak discrimination of gCJD E200K, gCJD V210I, and iCJD from and blurred. ADAM10 immunoreactivity was moderate in HC and ND showed a good diagnostic accuracy ranging neurons of the FC, weaker in the granular cells of the cer- from 0.78 to 0.90 (compared to HC) and from 0.73 to 0.85 ebellum and negative in Purkinje cells, in control and sCJD (compared to ND). In contrast, sTREM2 showed no diag- MM1 cases (Fig. 2c). sCJD VV2 was not analysed. Incuba- nostic value in distinguishing HC and ND from FFI cases tion with only the secondary antibody showed no immuno- (AUC 0.62 and 0.51, respectively) (Fig. 4b). sTREM2 was reactions (data not shown). also quantified in AD cases. Mean sTREM2 concentrations in AD were slightly higher than in HC and ND; however, Regional and temporal TREM2 expression differences did not reach statistical significance (Table 1 in the brain of sCJD mouse models and Fig. 4a). In contrast, sTREM2 levels were significantly higher in sCJD and gCJD-V210I compared to AD (Fig. 4a). To investigate the regional and temporal alterations of sTREM2 levels were also assessed in two series of MS cases TREM2 during prion pathogenesis in brain tissue, we using different populations and different methods. sTREM2 took advantage of two sCJD mouse models recapitulating levels in the CSF were not altered in MS when compared the molecular and pathological alterations of sCJD MM1 with controls (data not shown). (tg340-sCJD MM1) and sCJD VV2 (tg360-sCJD VV2) sub- Next, we explored the influence of codon 129 polymor- types [9, 10, 34]. Trem2 mRNA expression was investigated phism in PRNP on sTREM2 levels. The highest sTREM2 in the anterior and posterior cortex, cerebellum, olfactory concentrations were detected in sCJD cases harbouring bulb, and brainstem at pre-clinical (80 dpi), early clinical methionine/methionine (MM; 7884 ± 6652 pg/mL, n = 32), (120 dpi), and clinical (180 dpi) disease stages (n = 4–8) followed by methionine/valine (MV; 5257 ± 4061 pg/mL, (Fig. 3). At the clinical stage, Trem2 mRNA was increased n = 19) and valine/valine (VV; 4183 ± 3803 pg/mL, n = 17). in all brain regions in both CJD models. Increased Trem2 Statistical differences were detected between MM and expression was also observed at early-clinical stages in the VV cases (p = 0.03) (Fig. 5a). pROC analysis revealed a anterior cortex of both models. Differences between sCJD superior CSF sTREM2 diagnostic accuracy for sCJD MM models were detected in the posterior cortex, where Trem2 compared to sCJD VV cases, both when compared to HC was specifically increased at preclinical and early-clinical (AUC = 0.94 vs 0.82, p = 0.0177) and to ND (AUC = 0.90 stages in the tg340 sCJD model and in the cerebellum, where vs. 0.74, p = 0.0076) (Fig. 5a). Trem2 mRNA expression was specifically increased at early- We further evaluated CSF sTREM2 in the two most clinical stages in the tg361 sCJD model (Fig. 3). prevalent sCJD subtypes (MM1 and VV2) in those cases with prion type available from autopsy analysis. sCJD MM1 sTREM2 in the CSF of prion diseases (8196 ± 7782 pg/mL, n = 20) presented higher sTREM2 concentrations than sCJD VV2 cases (3224 ± 2200 pg/mL, The presence of increased TREM2 expression and ADAM10 n = 9) (Fig. 5b). In line with these observations, AUC val- expression and activation in the brain of sCJD raised the ues were significantly different between the two subtypes possibility that sTREM2 could be differentially altered in when compared to HC (AUC = 0.95 vs 0.81, p = 0.0151) and biological fluids of sCJD cases. CSF TREM2 concentra- ND (AUC = 0.90 vs. 0.73, p = 0.0070) (Fig. 5b), indicating tions were assessed in HC, ND, prion diseases from spo- a significantly higher diagnostic value of sTREM2 in the radic, genetic and acquired aetiology, AD, and MS (Table 1). discrimination of sCJD MM1 cases. Regarding genetic prion sTREM2 was associated neither with age at onset nor with diseases, gCJD-E200K and FFI cases were stratified accord- ing to their PRNP codon 129 genotype, which influences 13 850 Acta Neuropathologica (2021) 141:841–859 Fig. 3  mRNA Trem2 expression in the brain of sCJD MM1 and VV2 mouse models. RT-qPCR analysis Trem2 expression in mouse models of sCJD MM1 (tg340-CJD MM1) (left panels) and sCJD VV2 (tg361-CJD VV2) (right panels) subtypes. Trem2 expression was analysed in five brain regions: anterior and posterior cortex, cer- ebellum, olfactory bulb, and brainstem, at three time points: pre-clinical (80 days post inoculation (dpi)), early-clinical (120 dpi), and clinical (180 dpi). CJD-inoculated animals (black columns) were compared to their corresponding control- inoculated animals (white columns). Four to eight animals per time point and condition were used. Normalization was performed using the house- keeping gene Hprt; *p < 0.05, **p < 0.01 their clinicopathological features [17, 47]. sTREM2 con- nor between FFI-MM (n = 16) and FFI-MV (n = 10) cases centrations were not different between gCJD-E200K-MM (p = 0.61) (data not shown). (n = 33) and gCJD-E200K-MV (n = 22) cases (p = 0.59) Cerebrospinal fluid tests sTREM2 in sCJD displayed a positive correlation with t-tau (n = 126, cc = 0.3424, 13 Acta Neuropathologica (2021) 141:841–859 851 Fig. 4  CSF sTREM2 in the diagnosis of prion diseases. a CSF sTREM2 concentrations in HC (n = 48), ND (n = 64), sCJD (n = 139), gCJD-E220K (n = 57), gCJD-V210 (n = 34), FFI (n = 26), iCJD (n = 10), and AD (n = 35). b Area under the curve (AUC) derived from receiver operating character- istic (ROC) curves with 95% confidence interval (95% CI) for CSF sTREM2 in the discrimi- nation of prion diseases and AD from HC and ND; *p < 0.05, ***p < 0.001 p < 0.001) (Fig. 5c), and was associated with 14-3-3 posi- (p < 0.001) (Table 2, Fig. 6a), with an AUC value of 0.80 in tivity, since sCJD cases presenting a positive 14-3-3 test the discrimination of both diagnostic groups (Fig. 6b). (n = 118) showed higher sTREM2 concentrations than sTREM2 concentrations in sCJD were associated neither those with a negative (or inconclusive) 14-3-3 test (n = 21) with PRNP codon 129 genotype: MM (9755 ± 10,492 pg/ (p = 0.03) (Fig. 5d). Additionally, sTREM2 correlated with mL, n = 59), MV (8238 ± 4220 pg/mL, n = 8) and VV YKL-40 concentrations (n = 47, cc = 0.3240, p = 0.0263) (7787 ± 2773 pg/mL, n = 14) (p > 0.05 in all comparisons) (Fig. 5e). (Fig. 6c) nor with sCJD subtype: MM1 (10,040 ± 11,570, To investigate a potential influence of the disease stage n = 45) and VV2 cases (7106 ± 2147, n = 12) (p = 0.8654) at which the LP was performed and sTREM2 concentra- (Fig. 6d). In a subset of sCJD cases where plasma-CSF tions in sCJD patients, samples were stratified in early paired data were available (n = 31), sTREM2 showed a (7914 ± 5924 pg/mL, n = 9), middle (8229 ± 7854 pg/mL, significant correlation between the fluids (cc = 0.3671, n = 20), and late (6794 ± 4950 pg/mL, n = 35) stages, but p = 0.0422) (Fig. 6e). Plasma sTREM2 showed a mod- no significant differences were detected between groups est positive correlation with plasma YKL-40 (n = 63, (Fig. 5f). In 14 individuals, serial LPs at different disease cc = 0.2958, p = 0.0217) (Fig. 6f) Nfl (n = 40, cc: 0.4719, stages were available allowing investigation of whether p = 0.0021) (Fig. 6g) and t-tau (n = 40, cc = 0.3392, sTREM2 values increased during disease progression p = 0.0322) (Fig. 6h) concentrations. (Fig. 5g). Using a multilevel linear model, we observed a No significant differences in plasma sTREM2 were significant increase of 1962 pg/mL (95% CI 811–3113) per detected between the different disease stages at which blood disease stage (p = 0.0036). CSF sTREM2 concentrations was obtained: early (8258 ± 3842 pg/mL, n = 14), middle were not significantly associated with total disease duration (7852 ± 5371 pg/mL, n = 15), and late (9768 ± 10,699 pg/ (cc = − 0.1040, p = 0.2408) (Fig. 5h), ruling out a potential mL, n = 53) stages (p > 0.05 in all comparisons) (Fig. 6i). role of CSF sTREM2 as sCJD prognostic marker. Similar to CSF sTREM2, plasma sTREM2 was not associated with sCJD disease duration (cc = − 0.1053, sTREM2 in the plasma of sCJD p = 0.3463) (Fig. 6j). Soluble TREM2 concentrations in plasma have been reported to be in the same range as detected in the CSF Discussion [52]. Thus, we explored whether sTREM2 in plasma could be altered in sCJD compared to HC. Plasma sTREM2 in In the present study, we report TREM2 expression and HC and sCJD was associated neither with age at onset nor levels in the brain and biological fluids of prion diseases, with sex (Table 2). In sCJD, sTREM2 (8981 ± 8932 pg/mL) with special focus on sCJD, uncovering its potential role was significantly increased compared to HC (4409 ± 3961) as a diagnostic marker. In sCJD, we observed a significant increase in TREM2 mRNA compared with controls and 13 852 Acta Neuropathologica (2021) 141:841–859 Fig. 5  CSF sTREM2 in sCJD: association with codon129 genotype, ity. 14-3-3 positive cases; n = 118 and 14-3-3 negative cases; n = 21. sCJD subtype, CSF biomarkers, and clinical data. a CSF sTREM2 Mean ( +), median (horizontal bar inside the box), and interquartile concentrations stratified by prion protein gene (PRNP) codon 129 ranges are represented. e Correlation analysis between CSF sTREM2 polymorphism (M = methionine, V = valine). sCJD MM; n = 32, and CSF YKL-40 (n = 47). f CSF sTREM2 concentrations stratified sCJD MV; n = 19 and sCJD VV; n = 17. Mean ( +), median (horizon- by disease stage at the time of lumbar puncture (LP). Samples were tal bar inside the box) and interquartile ranges are represented. Area grouped into three categories according to whether they underwent under the curve (AUC) values with 95% confidence intervals (95% LP in the first (< 0.33), second (0.33–0.66) or third (> 0.66) stage of CI) indicated for each comparison. b CSF sTREM2 concentrations the disease. Early disease stage, n = 15; middle disease stage, n = 21; stratified by sCJD subtype MM1 (n = 20) and VV2 (n = 9). Mean late disease stage, n = 60. Mean ( +), median (horizontal bar inside ( +), median (horizontal bar inside the box), and interquartile ranges the box), and interquartile ranges are represented. g Longitudinal are represented. Area under the curve (AUC) values with 95% confi- analysis of CSF TREM2 concentrations in cases with available LP dence intervals (95% CI) indicated for each comparison. c Correlation at different disease stages (n = 14). h Correlation analysis between analysis between CSF TREM2 and CSF t-tau (n = 126). d Associa- sTREM2 concentrations and disease duration (months) (n = 126). cc: tion analysis of CSF TREM2 concentrations with CSF 14-3-3 positiv- correlation coefficient; *p < 0.05, **p < 0.01, ***p < 0.001 13 Acta Neuropathologica (2021) 141:841–859 853 Fig. 6  Plasma sTREM2 in sCJD: association with codon129 genotype, sCJD sub- type, plasma biomarkers, and clinical data. a Plasma sTREM2 concentrations in HC (n = 97) and sCJD (n = 86) cases. b Area under the curve (AUC) derived from receiver operating char- acteristic (ROC) curves with 95% confidence interval (95% CI) for plasma sTREM2 in the discrimination of sCJD from HC. c Plasma sTREM2 con- centrations stratified by prion protein gene (PRNP) codon 129 polymorphism (M = methionine, V = valine). sCJD MM; n = 59, sCJD MV; n = 8 and sCJD VV; n = 14. Mean ( +), median (horizontal bar inside the box), and interquartile ranges are represented. d Plasma sTREM2 concentrations stratified by sCJD subtype MM1 (n = 45) and VV2 (n = 12). Mean ( +), median (horizontal bar inside the box) and interquartile ranges are represented. e Correlation analysis between plasma and CSF sTREM2 (n = 31). Correla- tion analysis between plasma sTREM2 and plasma YKL-40 f (n = 63), plasma Nfl (n = 40) g and plasma t-tau (n = 40) h. i Plasma sTREM2 concentrations stratified by disease stage at the time of blood uptake. Samples were grouped into three catego- ries according to whether they underwent blood uptake in the first (< 0.33), second (0.33– 0.66), or third (> 0.66) stage of the disease. Early disease stage, n = 14; middle disease stage, n = 15; late disease stage, n = 53. Mean ( +), median (horizontal bar inside the box), and inter- quartile ranges are represented. j Correlation analysis between plasma sTREM2 concentrations and disease duration (months) (n = 82). cc: correlation coef- ficient; ***p < 0.001 13 854 Acta Neuropathologica (2021) 141:841–859 FTLD-TDP43. TREM2 protein is increased in the FC and astrocytic marker YKL-40. Similarly, plasma sTREM2 posi- CB of sCJD post-mortem-human brain tissue at the protein tively correlates with YKL-40, t-tau, and Nfl. The moder- level (both in its immature and mature forms), as revealed ate but significantly positive association between CSF and with western blotting. Immunohistochemistry using the plasma sTREM2 in sCJD cases, similar to what is observed anti-TREM2 antibody from RD Systems (AF1828) shows in AD [4], suggests that plasma sTREM2 may reflect the TREM2 expression in blood vessels and microglia. Neurons CNS neuroinflammatory profile in these conditions. How- are also stained using goat anti-TREM2 Abcam antibody ever, the reported expression of TREM2 in non-CNS tis- in our cases. TREM2 immunoreactivity has been reported sues [14, 74], especially in pathological conditions, does not in neurons, astrocytes, and oligodendrocytes using goat rule out a potential contribution of these tissues to plasma anti-TREM2 Abcam antibody in another study [11]. Since sTREM2 concentrations and could explain the differing there is no evidence of neuronal TREM2 mRNA produc- influence of PRNP codon 129 and sCJD subtype between tion in neurons, and the Abcam antibody is discontinued, CSF and plasma. we assume that additional studies are needed to postulate The influence of PRNP codon 129 on CSF sTREM2 TREM2 as a neuronal marker. (higher concentrations in MM compared to VV cases) is The positive correlation between TREM2 and the mac- different to that reported for neuronal damage markers such rophage marker CD68 at the mRNA level supports immuno- as CSF t-tau and 14-3-3, which display significantly lower histochemical findings and suggests that microglia account values in CJD MV cases compared to homozygotes [19, 27], for the increased TREM2 expression in sCJD brain. This and is explained by the less severe phenotype and longer observation, in conjunction with increased ADAM10 disease duration of CJD MV cases. expression and activation, points to a potential increase of Importantly, the increased CSF TREM2 concentrations in sTREM2 in biological fluids of prion diseased patients. sCJD MM compared to sCJD VV cases led to significantly In agreement with this hypothesis, sTREM2 dis- better AUC values in MM cases, reaching high discrimina- plays good accuracy in discriminating sCJD from ND tory power compared to controls (HC, AUC = 0.94 and ND, (AUC = 0.85) and HC (AUC = 0.90) in CSF, and from HC AUC = 0.90). Thus, data on PRNP polymorphism (routine (AUC = 0.80) in plasma. This despite diagnostic accuracies test in prion diagnostic centres) may improve the accuracy being lower than those reported for CSF biomarkers cur- of CSF sTREM2 in terms of disease diagnosis and should be rently used in clinical settings (14-3-3, t-tau (p-tau/t-tau) and taken into consideration in patient stratification when used as RT-QuIC) [13, 36, 44, 62]. The CSF sTREM2 increase in a potential surrogate endpoint in disease-modifying therapy. gCJD and iCJD is in line with the similar clinicopathologi- The reason for increased CSF sTREM2 in sCJD MM1 cal features of both conditions with sCJD, and consequently, compared to sCJD VV2 remains unknown, but it could with their fluid biomarkers profiles [32, 38]. In contrast, CSF be explained by the higher microglial profile in the corti- sTREM2 is not altered in FFI, in agreement with the low cal regions of sCJD MM1 compared with VV2 cases [34]. sensitivity of prion disease biomarkers in this condition [40], This, together with the observation that increased TREM2 most likely reflecting the restricted pathological alterations expression is more significant in FC than in CB, suggests in terms of neuroinflammation and longer disease duration that sTREM2 derived from cortical regions is the main con- compared with CJD cases [8]. tributor to its increased concentrations in biological fluids. Previous studies have shown slightly or moderately Another interesting finding in our study is the obser- increased sTREM2 levels in the CSF in patients with AD vation that sCJD mouse models have increased TREM2 [22, 24, 55, 67, 68]. Moreover, individuals with MS and levels, occurring in some brain regions at pre-clinical and other inflammatory CNS diseases show increased CSF early-clinical stages in a regional and disease subtype-spe- sTREM levels [1, 48, 78]. However, we have not found sig- cific manner. This is in line with the observation that CSF nificant differences in the CSF levels of sTREM2 in AD and sTREM2 concentrations are sCJD-subtype-specific and MS cases in our series. We can interpret these discrepancies independent of the disease stage at which sampling is per- due to lower discrimination capacity of our methods when formed. It is therefore tempting to speculate that sTREM2 compared with those used in other labs. If this hypothesis is alterations in biological fluids may occur at early disease true, the present findings reinforce the magnitude of sTREM stages. Unfortunately, the absence of a prodromal stage of alterations in prion diseases when compared with AD and sCJD precludes the study of pre-clinical cases and leaves MS. this possibility open for the analysis of sTREM2 in asymp- Interestingly, and in contrast to TREM2, sCJD neuroax- tomatic gCJD cases associated with the E200K and V210I onal damage markers with exclusive neuronal expression mutations. such as t-tau and 14-3-3 display reduced protein levels in In contrast with longitudinal data from neuroaxonal/syn- sCJD brain tissue [39]. CSF sTREM2 correlates with the aptic damage markers such as t-tau, 14-3-3, Nfl, and α-syn, neuronal damage markers t-tau and 14-3-3 and with the which present marginal or absent alterations of their CSF 13 Acta Neuropathologica (2021) 141:841–859 855 concentrations along disease progression [33, 59, 77], the Alberta. JADR was supported by grants from the Spanish Ministry of progressive neuroinflammatory profile in sCJD brain may Science, Innovation and Universities (MICINN/FEDER) (RTI2018- 099773-B-I00), the CERCA Programme, and by the Commission for explain increased CSF sTREM2 concentrations in follow-up Universities and Research of the Department of Innovation, Universi- LPs, suggesting a potential role for sTREM2 as a candidate ties, and Enterprise of the Generalitat de Catalunya (SGR2017-648) marker of microglial activation. The observation that CSF and CIBERNED (CMED2018-2). and plasma sTREM2 are not associated with disease dura- tion in sCJD precludes a prognostic value for sTREM2. Author contributions FL designed the study. DD-L, NK, AV-P, OL-P, KT, MS, JEC, PA-B, HP, and VS performed the experiments and In conclusion, we have found expression of TREM2 in a analysed the data. PH, AL, MC, JR, HZ, KB, JAR, RS-V, BM, IF, subpopulation of microglial cells in human sCJD brains, with IZ, and FL contributed to clinical and biochemical data acquisition, increased mRNA and protein levels in brain compared to con- interpretation, and sampling. AM-M, JCE, and JMT performed animal trols. Further, we have shown that CSF and plasma sTREM2 experimentation. EV contributed to technical and analytical expertise. FL, IF and IZ drafted the manuscript; all authors critically revised the have diagnostic accuracy in the discrimination of sCJD. We manuscript and approved its contents before submission. may speculate that sTREM2 quantification in CSF may be useful for complementary diagnostic purposes. However, the Funding We thank the HUB-ICO-IDIBELL-Biobank and the CERCA assessment of TREM2 levels in the CSF in other neurological Programme of the Generalitat de Catalunya for institutional support. diseases, that may represent clinical diagnosis difficulties with This study was funded by the Instituto Carlos III (Grants Number CP16/00041 and PI19/00144) to FL. This project was also funded by CJD, is a pending condition. Unfortunately, adequate numbers la Fundació La Marató de TV3 (Grants No. 201821-30, 201821-31 of samples from cases with paraneoplastic encephalitis, other and 201821-32 to FL, JCE and EV, respectively) and by the Fondo rare encephalitis, and rapid forms of non-prionic dementia Europeo de Desarrollo Regional (FEDER) through the Interreg V-A were not available in the present study. Finally, our data high- España-Francia-Andorra (POCTEFA 2014–2020) programme (at 65%) to IF. AVP is supported by the Beatriu de Pinós programme light the importance of molecular neuropathology assessment (2018-BP-00129) from the Ministry of Business and Knowledge of in the context of biomarker research toward understanding the Government of Catalonia, and cofunded by the EU Horizon 2020 of the aetiology of the alterations detected in biological flu- programme under an MSCA grant agreement (801370). HZ is a Wal- ids, which in turn may be relevant in the interpretation of the lenberg Scholar supported by grants from the Swedish Research Coun- cil (#2018-02532), the European Research Council (#681712), Swedish translational value of biomarker profiles. State Support for Clinical Research (#ALFGBG-720931), the Alzhei- mer Drug Discovery Foundation (ADDF), USA (#201809-2016862), Supplementary Information The online version contains supplemen- and the UK Dementia Research Institute at UCL. KB is supported by tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00401-0​ 21-0​ 2296-1. the Swedish Research Council (#2017-00915), the Alzheimer Drug Discovery Foundation (ADDF), USA (#RDAPB-201809-2016615), the Acknowledgements We thank the HUB-ICO-IDIBELL-Biobank and Swedish Alzheimer Foundation (#AF-742881), Hjärnfonden, Sweden the CERCA Programme of the Generalitat de Catalunya for institu- (#FO2017-0243), the Swedish state under the agreement between the tional support. This study was funded by the Instituto Carlos III (Grants Swedish government and the County Councils, the ALF-agreement Number CP16/00041 and PI19/00144) to FL. This project was also (#ALFGBG-715986), and the European Union Joint Programme for funded by la Fundació La Marató de TV3 (Grants No. 201821-30, Neurodegenerative Disorders (JPND2019-466-236). AL is supported 201821-31 and 201821-32 to FL, JCE and EV, respectively) and co- by the Ministero della Salute, Italy, for the national surveillance of financed by ERDF under the programme Interreg Poctefa: RedPrion Creutzfeldt-Jakob disease. This research was also supported in part by 148/16 to IF. AVP is supported by the Beatriu de Pinós programme the Alberta Synergies in Alzheimer’s and Related Disorders (SynAD) (2018-BP-00129) from the Ministry of Business and Knowledge of programme which is funded by the Alzheimer Society of Alberta and the Government of Catalonia, and cofunded by the EU Horizon 2020 Northwest Territories through their Hope for Tomorrow programme programme under an MSCA grant agreement (801370). HZ is a Wal- and the University Hospital Foundation. SynAD operates in partnership lenberg Scholar supported by grants from the Swedish Research Coun- with the Neuroscience and Mental Health Institute at the University of cil (#2018-02532), the European Research Council (#681712), Swedish Alberta. JADR was supported by grants from the Spanish Ministry of State Support for Clinical Research (#ALFGBG-720931), the Alzhei- Science, Innovation and Universities (MICINN/FEDER) (RTI2018- mer Drug Discovery Foundation (ADDF), USA (#201809-2016862), 099773-B-I00), the CERCA Programme, and by the Commission for and the UK Dementia Research Institute at UCL. KB is supported by Universities and Research of the Department of Innovation, Universi- the Swedish Research Council (#2017-00915), the Alzheimer Drug ties, and Enterprise of the Generalitat de Catalunya (SGR2017-648) Discovery Foundation (ADDF), USA (#RDAPB-201809-2016615), the and CIBERNED (CMED2018-2). Swedish Alzheimer Foundation (#AF-742881), Hjärnfonden, Sweden (#FO2017-0243), the Swedish state under the agreement between the Declarations Swedish government and the County Councils, the ALF-agreement (#ALFGBG-715986), and the European Union Joint Programme for Neurodegenerative Disorders (JPND2019-466-236). AL is supported Conflict of interest HZ has served on scientific advisory boards for by the Ministero della Salute, Italy, for the national surveillance of Denali, Roche Diagnostics, Wave, Samumed, Siemens Healthineers, Creutzfeldt-Jakob disease. This research was also supported in part by Pinteon Therapeutics, and CogRx, has given lectures in symposia the Alberta Synergies in Alzheimer’s and Related Disorders (SynAD) sponsored by Fujirebio, Alzecure, and Biogen, and is a co-founder of programme which is funded by the Alzheimer Society of Alberta and Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part Northwest Territories through their Hope for Tomorrow programme of the GU Ventures Incubator Programme. KB has served as a con- and the University Hospital Foundation. SynAD operates in partnership sultant, on advisory boards, and on data monitoring committees for with the Neuroscience and Mental Health Institute at the University of Abcam, Axon, Biogen, Julius Clinical, Lilly, MagQu, Novartis, Roche 13 856 Acta Neuropathologica (2021) 141:841–859 Diagnostics, and Siemens Healthineers, and is a co-founder of Brain 4. 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MBio 11:1–19. https://​doi.​org/​10.​ ioural Research with Animals (Directive 2010/63/EU) and made every 1128/​mBio.​00393-​20 effort to minimize suffering. Experiments developed in the Centro de 10. Cassard H, Torres JM, Lacroux C, Douet JY, Benestad SL, Lantier Investigación en Sanidad Animal–Instituto Nacional de Investigación F et al (2014) Evidence for zoonotic potential of ovine scrapie y Tecnología Agraria y Alimentaria (Madrid, Spain) were evaluated prions. Nat Commun 5(5):5821. https://​doi.​org/​10.​1038/​ncomm​ by the Committee on the Ethics of Animal Experiments of the Insti- s6821 tuto Nacional de Investigación y Tecnología Agraria y Alimentaria 11. Chen S, Peng J, Sherchan P, Ma Y, Xiang S, Yan F et al (2020) and approved by the General Directorate of the Madrid Community TREM2 activation attenuates neuroinflammation and neuronal Government (permit no. PROEX 094/18). apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice. J Neuroinflammation 17(1):168. https://​doi.​org/​10.​1186/​ s12974-​020-​01853-x Open Access This article is licensed under a Creative Commons Attri- 12. Cheng-Hathaway PJ, Reed-Geaghan EG, Jay TR, Casali BT, bution 4.0 International License, which permits use, sharing, adapta- Bemiller SM, Puntambekar SS et al (2018) The Trem2 R47H tion, distribution and reproduction in any medium or format, as long variant confers loss-of-function-like phenotypes in Alzheimer’s as you give appropriate credit to the original author(s) and the source, disease. Mol Neurodegener 13:29. https://​d oi.​o rg/​1 0.​1 186/​ provide a link to the Creative Commons licence, and indicate if changes s13024-​018-​0262-8 were made. The images or other third party material in this article are 13. 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P et al (2015) Triggering receptor expressed on myeloid cells-2 is involved in prion-induced microglial activation but does not Authors and Affiliations Daniela Diaz‑Lucena1,2 · Niels Kruse3 · Katrin Thüne4 · Matthias Schmitz4,5 · Anna Villar‑Piqué1,2 · Jose Eriton Gomes da Cunha6 · Peter Hermann4 · Óscar López‑Pérez1,2 · Pol Andrés‑Benito1,2 · Anna Ladogana7 · Miguel Calero8,9 · Enric Vidal10 · Joachim Riggert11 · Hailey Pineau12 · Valerie Sim12 · Henrik Zetterberg13,14,15,16 · Kaj Blennow13,14 · Jose Antonio del Río17,18,19,20 · Alba Marín‑Moreno21 · Juan Carlos Espinosa21 · Juan María Torres21 · Raquel Sánchez‑Valle22 · Brit Mollenhauer23,24 · Isidre Ferrer1,2,25 · Inga Zerr4,5 · Franc Llorens1,2,4 1 14 Network Center for Biomedical Research Clinical Neurochemistry Laboratory, Sahlgrenska University in Neurodegenerative Diseases (CIBERNED), Hospital, Mölndal, Sweden L’Hospitalet de Llobregat, Spain 15 Department of Neurodegenerative Disease, UCL Institute 2 Bellvitge Biomedical Research Institute (IDIBELL), of Neurology, London, UK L’Hospitalet de Llobregat, Spain 16 UK Dementia Research Institute, London, UK 3 University Medical Center Göttingen, Institute 17 Molecular and Cellular Neurobiotechnology, Scientific of Neuropathology, Göttingen, Germany Park of Barcelona, Institute for Bioengineering of Catalonia 4 Department of Neurology, University Medical Center (IBEC), The Barcelona Institute for Science and Technology Göttingen, Gern August University, Robert Koch Strasse 40, (BIST), Barcelona, Spain 37075 Göttingen, Germany 18 Department of Cell Biology, Physiology and Immunology, 5 German Center for Neurodegenerative Diseases (DZNE), Faculty of Biology, University of Barcelona, Barcelona, Göttingen, Germany Spain 6 19 Keizo Asami Laboratory (LIKA), Universidade Federal de Network Center for Biomedical Research Pernambuco (UFPE), Recife, Brazil in Neurodegenerative Diseases (CIBERNED), Barcelona, 7 Spain Department of Neurosciences, Istituto Superiore Di Sanità, 20 Rome, Italy University of Barcelona, Institute of Neuroscience, 8 Barcelona, Spain Alzheimer Disease Research Unit, CIEN Foundation, 21 Chronic Disease Programme, Queen Sofia Foundation Centro de Investigación en Sanidad Animal, CISA-INIA, Alzheimer Center, Instituto de Salud Carlos III, Madrid, Madrid, Spain Spain 22 Alzheimer’s Disease and Other Cognitive Disorders Unit, 9 Network Center for Biomedical Research Neurology Department, Hospital Clinic de Barcelona, in Neurodegenerative Diseases (CIBERNED), Madrid, Spain Institut D’Investigacions Biomediques August Pi I Sunyer 10 (IDIBAPS), Barcelona, Spain Centre de Recerca en Sanitat Animal, Campus Universitat 23 Autònoma de Barcelona, Institut de Recerca I Tecnologia Paracelsus-Elena Klinik, Kassel, Germany Agroalimentàries, Bellaterra, Spain 24 Department of Neurology, University Medical Centre 11 Department of Transfusion Medicine, University Medical Göttingen, Göttingen, Germany School, Göttingen, Germany 25 Department of Pathology and Experimental Therapeutics, 12 Department of Medicine‑Division of Neurology, Centre Hospitalet de Llobregat, University of Barcelona, Feixa for Prions and Protein Folding Diseases, University Llarga S/N, 08907 Barcelona, Spain of Alberta, Edmonton, Canada 13 Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at the University of Gothenburg, Institute of Neuroscience and Physiology, Mölndal, Sweden 13