Thanks for visiting The use of software that blocks ads hinders our ability to serve you the content you came here to enjoy We ask that you consider turning off your ad blocker so we can deliver you the best experience possible while you are here Please enable JS and disable any ad blocker Please select what you would like included for printing: Copy the text below and then paste that into your favorite email application where she was also a member of the Rosary Society Elizabeth was employed by General Electric She enjoyed working with her hands sewing and doing needlework Elizabeth was very talented at baking and canning for her family she enjoyed watching the Chicago Cubs and I.U Indiana; 12 grandchildren; 25 great-grandchildren; and 27 great-great-grandchildren Elizabeth was preceded in death by her two grandsons A Mass of Christian Burial will be offered at 10:00 a.m Mary of the Assumption Catholic Church with Father David Ruppert officiating Burial will follow in the Decatur Cemetery.  The family will receive friends from 2:00 to 7:00 p.m at the Zwick & Jahn Funeral Home in Decatur and one hour before the service at the church on Tuesday.  The Holy Rosary will be recited at 7:00 p.m Preferred memorials can be given to ACCF – Cancer Fund Arrangements by Zwick and Jahn Funeral Homes of Decatur Enter your phone number above to have directions sent via text This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply Service map data © OpenStreetMap contributors four times as much green electricity: RWE has successfully completed the modernisation of its wind turbine in Lengerich the old 1.8 megawatt (MW) turbine was replaced by a more powerful one The new 5.7 MW turbine at the Emsland location in north-west Germany now supplies around 4,000 instead of 1,000 households with green electricity A total of around 50 employees from RWE and various partner companies were onsite throughout the construction period They ensured that everything went smoothly RWE’s head of construction onshore wind and solar in Germany: “Lengerich is a showcase project for us The old plant has been reliably generating wind power for more than 20 years Technological progress meant it was time to move on to a new generation To plan the delivery of the 80-metre-long rotor blades we optimised the route in advance using a 2D simulation programme That enabled us to avoid bottlenecks along the route from the factory in Rostock to Lower Saxony we used a special trailer whose axles can be controlled independently of the towing vehicle when cornering Thanks to everyone involved for their great work.” The surrounding municipalities also benefit from the repowering of the Lengerich wind turbine RWE voluntarily pays 0.2 cents to the neighbouring communities for every kilowatt hour produced they can expect a fourfold increase in income to around €28,000 per year compared to €7,000 without repowering RWE increases output of existing wind farms RWE has recently commissioned another repowering project in North Rhine-Westphalia the new wind turbines at the Elisenhof wind farm generate enough green electricity to supply 5,500 households per year The dismantled main components of the old wind farm were overhauled in the company’s own workshop and will now extend the service life of identical RWE wind turbines in Spain by 10 to 15 years RWE will repower further wind farms in Lower Saxony and Schleswig-Holstein increasing the total capacity from around 41 MW to over 84 MW The company currently operates around 90 wind farms in Germany – and the number is growing Images for media use of the Lengerich wind turbine are available at the are available at the media centre Metrics details Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer’s disease (AD) suggesting that activation of this innate immune receptor may be a useful therapeutic strategy Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site to facilitate blood–brain barrier transcytosis ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody In human induced pluripotent stem cell (iPSC)-derived microglia ATV:TREM2 induced proliferation and improved mitochondrial metabolism Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states which were distinct from those induced by amyloid pathology ATV:TREM2 boosted brain microglial activity and glucose metabolism ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD Microglia are the brain’s resident innate immune cells that can resolve disturbances and help maintain brain homeostasis these studies suggest that TREM2 loss of function (LOF) contributes to AD risk and increased TREM2 function may be beneficial in AD ATV:TREM2 demonstrated improved brain exposure and CNS biodistribution compared to anti-TREM2 ATV:TREM2 promoted mitochondrial metabolic pathways such as lipid catabolism and glucose oxidation in microglia ATV:TREM2 induced transcription of metabolic pathway genes and increased brain glucose uptake by fluorodeoxyglucose-positron emission tomography (FDG-PET) These studies provide new insights into the mechanisms by which ATV:TREM2 Five distinct clusters of microglia were identified Stacked bar plots of clusters distributed per group for both studies Clusters are shown as percentages of the whole microglial compartment averaged for each biological replicate UMAP projection split by group for cluster distribution Data from WT;TfRmu/hu mice are boxed with solid lines and data from AppSAA;TfRmu/hu mice are boxed with dashed lines Heat map of average log2FC in each cluster compared to the ‘homeostatic’ cluster Pseudobulk expression was generated by summing every counts per gene Each mouse was treated as a biological replicate and differential expression (DE) was performed for each cluster versus ‘homeostatic’ using limma Scatter plots of the comparison of versus ‘homeostatic’ log2FC for every gene between clusters 3 and 2 (top) and clusters 3 and 5 (bottom) genes falling below the dashed line are more upregulated in cluster 3 whereas genes above the dashed line are further upregulated in cluster 2 (top) or cluster 5 (bottom) Canonical DAM genes and other genes of interest are highlighted in orange Dot plot showing GSEA for each cluster using DE versus ‘homeostatic.’ Signatures were taken from the hallmark gene signatures collection Dot size is inversely proportional to log10(corrected P value) Source data ATV enhances microglia responses of a TREM2 antibody despite matching brain exposures for anti-TREM2 ATV:4D9 induced dynamic microglial states transcriptionally and morphologically Antibody schematic of ATV:TREM2 with human TREM2 Fab affinity and ATV binding site in the Fc domain and effectorless Fc mutations Fluorescence polarization (FP)-based detection of TREM2 stalk peptide cleavage by recombinant ADAM17 (n = 4 independent experiments; two-tailed unpaired t-test ATV:TREM2 and PS liposome co-treatment enhanced pSyk in WT iMG (n = 3 independent experiments; Tukey’s multiple comparisons test Schematic illustrating ATV and TREM2 Fab valency effects on pSyk signaling Antibodies include anti-TREM2 and ATV:TREM2 with MV and BV Fabs hTREM2-DAP12 HEK293 cells treated with a dose response of antibodies for 5 minutes followed by pSyk detection (n = 3 independent experiments; mean ± s.e.m.) pSyk is blocked by co-treatment of ATV:TREM2 and anti-TfR hTREM2-DAP12 HEK293 cells were dosed with 100 nM TREM2 antibodies and a titration anti-TfR pSyk was detetced 5 minutes after treatment (n = 3 independent experiments; mean ± s.e.m.) hTREM2-DAP12 HEK293 cells were treated with 100 nM per antibody for 5 minutes followed by IP on cell lysates with anti-TREM2 Co-IP quantification of western blot data in g (n = 6 independent experiments; Wilcoxon test for ISO versus anti-TREM2 two-tailed paired t-test for ATV:ISO versus ATV:TREM2 Schematic of BioID TREM2 receptor clustering assay strategy Representative western blot detection of biotinylated TREM2 after streptavidin IP TREM2-BioID expression was induced 24 hours before the assay with 2 ng ml−1 of Dox Cells were treated with 100 nM antibody and 2 µM biotin Quantification of western blot from j (n = 4 independent experiments; ratio of two-tailed paired t-test Representative immunofluorescence images of hTREM2-DAP12 HEK293 cells stained for IgG (green) Cells were treated with 10 nM per antibody for 10 min Quantification of spot intensity for IgG and pSyk immunofluorescence per cell (n = 3 independent experiments with 3,000–5,000 cells per condition; Tukey’s multiple comparisons test Quantification of percent of IgG or pSyk spots localized within EEA1+ endosomes (n = 3 independent experiments with 3,000–5,000 cells per condition; Tukey’s multiple comparisons test Source data these data show that ATV:TREM2 does not block lipid ligands and signals sufficiently to promote cellular function these studies indicate that ATV:TREM2 mediates TfR–TREM2 binding in cis to enhance TREM2 signaling These data suggest that ATV:TREM2 enhances TREM2 signaling not solely by increasing bound antibody but likely due to TfR-mediated intracellular trafficking ATV:TREM2 becomes localized to endosomes with pSyk These studies demonstrate dual mechanisms of action in which ATV enhances TREM2 antibody function via TfR–TREM2 clustering and endosomal signaling These studies elucidate mechanisms of action for ATV:TREM2 mediated by TfR engagement Representative western blot images for p-mTOR (S2448) pGSK3b (S9) and pRPS6 (S235/236) in WT iMG treated with 100 nM ATV:TREM2 or isotype control RPS6-S235/236 (d) and GSK3b-S9 (e) phosphorylation levels were normalized to actin Relative expression was calculated by normalizing to vehicle control (PBS) for each experiment (n = 10 independent experiments (b–d) and n = 9 independent experiments (e); two-tailed paired t-test ATV:TREM2 induces proliferation in WT iMG but not TREM2 KO iMG WT or TREM2 KO iMG were treated with 100 nM ATV:TREM2 or isotype control for 96 hours The proliferation index was calculated as percentage of EdU+ cells normalized to vehicle control (PBS) (n = 3 independent experiments; two-tailed multiple-paired t-test Quantification of WT and PLCG2 KO iMG proliferation iMG were treated with 100 nM ATV:TREM2 or ATV:ISO (n = 4 independent experiments (WT) and n = 3 independent experiments (TREM2 KO); two-tailed multiple-paired t-test Representative images of WT iMG proliferation Cells were treated with vehicle (DMSO) or mTOR inhibitor AZD8055 (AZD) Quantification of WT iMG proliferation treated with mTOR inhibitor AZD8055 20 nM AZD8055 was co-dosed with 100 nM ATV:TREM2 for 96 hours (n = 5 independent experiments (DMSO) and n = 4 independent experiments (AZD); two-tailed multiple-paired t-test Nuclei quantification of iMG co-treated with ATV:TREM2 and AZD Relative nuclei count was normalized to vehicle control (PBS) for each experiment (n = 5 independent experiments (DMSO) and n = 4 independent experiments (AZD); two-tailed unpaired t-test RNA-seq of iMG treated for 4 days with PBS 100 nM ATV:ISO or ATV:TREM2 or 10 ng ml−1 LPS Relative expression (z-scores) of the top-most upregulated or downregulated genes selected from pathways of interest Pathway definitions were taken from the hallmark MSigDB collection; genes shown are a subset of those found in the leading edge of the gene set for each category Source data These data show that ATV:TREM2 promotes microglia proliferation via mTOR and PLCG2 and is biologically distinct from other innate immune stimuli ELISA detection of antibodies in whole brain lysates for mice dosed with ATV:TREM2 (1 10 or 30 mg kg−1) and anti-TREM2 (30 mg kg−1) 1 day after dose (n = 5 mice per group) Microglia detected by IBA1 staining (purple) and proliferative cells were detected by EdU labeling (green) 4 days after dose Quantification of IBA1/EdU staining (n = 5 mice per group; Dunnett’s multiple comparisons test CSF1R detected by ELISA in whole brain lysate (n = 5 mice per group; Kruskal–Wallis test for day 1 and Dunnett’s multiple comparisons test for day 4) CSF1R detected by ELISA in CSF (n = 5 mice per group for day 1 (30 mg kg−1 ATV:ISO); day 1 (3 mg kg−1 ATV:TREM2); day 1 (10 mg kg−1 ATV:TREM2); day 1 (30 mg kg−1 ATV:TREM2); day 4 (30 mg kg−1 ATV:ISO) n = 4 mice per group for rest and Dunnett’s multiple comparisons test for day 1; Kruskal–Wallis test for day 4 Ex vivo microglial phagocytosis for different substrates Myelin debris (f) (n = 8 mice per group; Dunnett’s multiple comparisons test) and Aβ (g) (n = 8 mice per group; two-tailed unpaired t-test whereas ATV110nM:TREM2 induced similar CSF1R levels as ATV1100nM:TREM2 suggesting that ATV requires an affinity threshold to enhance TREM2 Fab activity SPECT imaging showed that ATV:TREM2 significantly improved brain biodistribution compared to anti-TREM2 these results demonstrate that ATV:TREM2 is broadly distributed throughout the brain and acute TREM2 activation is sufficient to mediate sustained microglial responses as effects persist after antibody clearance these data suggest that ATV:4D9 biodistribution is largely driven by TfR rather than TREM2 binding thymus and sciatic nerve when compared to vehicle control in a 12-week study of ATV:4D9 in WT;TfRmu/hu mice dosed weekly at 10 mg kg−1 Representative microscopy images from iMG treated with 10 µM oleic acid and then 100 nM ATV:ISO or ATV:TREM2 for 48 hours BODIPY fluorescence (shown in green) was quantified (n = 5 independent experiments; two-tailed paired t-test Heat map of LC–MS analysis for TG species and acyl carnitines modulated by ATV:TREM2 in iMG treated with myelin Plotted values are log2-transformed raw counts and scaled by row ATV:TREM2 reduces TGs and increases acyl carnitines in WT iMG treated with myelin (c) (n = 6 independent experiments (TG) and n = 3 independent experiments (acyl carnitines); two-tailed paired t-test mean ± s.e.m.) but not PLCG2 KO iMG treated with myelin (d) (n = 3 independent experiments; two-tailed paired t-test Seahorse fatty acid oxidation OCR respiration measurements in TREM2 KO and PLCG2 KO iMG (n = 3 independent experiments; mean ± s.e.m.) ATV:TREM2 increases maximal respiration in WT iMG detected with Seahorse fatty acid oxidation OCR measurements (n = 7 independent experiments; two-tailed paired t-test The CPT-1 inhibitor etomoxir blocks the effect of ATV:TREM2 on respiration (n = 5 independent experiments; two-tailed paired t-test ATV:TREM2 was treated at 100 nM for 3 days The ATV:TREM2 effect is blocked by an MPC inhibitor UK5099 (n = 4 independent experiments; two-tailed paired t-test ATV:TREM2 increases average TMRE intensity in iMG after 3 days with 100 nM antibody (n = 6 independent experiments; two-tailed paired t-test Representative images of super-resolution microscopy of TMRE staining in iMG Mitochondria were segmented into networked and punctate morphologies Morphometric analysis of the prevalence of networked mitochondria (n = 3 independent experiments Volcano plots of RNA-seq analysis of microglia isolated from mice dosed with 10 mg kg−1 of ATV:ISO or ATV:TREM2 for 1 day Red or blue indicate significantly upregulated or downregulated genes The x axis represents log2 fold change in expression compared to vehicle-treated mice and the y axis represents –log10 adjusted P value Relative expression (z-scores) of the top-most upregulated or downregulated genes at day 1 after dose selected from oxidative phosphorylation and glycolysis pathways these results indicate that ATV:TREM2 increases the energetic capacity of microglia by promoting fatty acid oxidation and aerobic respiration via glucose catabolism consistent with ATV:TREM2ʼs ability to boost microglial respiration TSPO-PET imaging could be an indicator of ATV:TREM2 function in vivo Coronal and axial slices show cold scaled group average images of TSPO-PET (SUVH) projected upon a standard MRI T1-weighted atlas from 5×FAD;hTREM2 tg;TfRmu/hu mice (top row) or WT;hTREM2 tg;TfRmu/hu mice (bottom row) mice dosed with 10 mg kg−1 of antibody and 8 days after dose in 5×FAD;hTREM2 tg;TfRmu/hu mice (b) and WT;hTREM2 tg;TfRmu/hu mice (c) Scatter plot of individual TSPO-PET (SUVH) values Dotted lines represent linear associations between interval after antibody dosing and TSPO-PET quantification per group and with a 95% confidence interval (n = 6 mice per group; two-tailed unpaired t-test for each timepoint which used the two-tailed unpaired t-test with Welch’s correction) Coronal and axial slices show cold scaled group average images of FDG (SUV) projected upon a standard MRI T1-weighted atlas from 5×FAD;hTREM2 tg;TfRmu/hu mice (top row) or WT;hTREM2 tg;TfRmu/hu mice (bottom row) after 10 mg kg−1 of antibody Quantification of cortical glucose uptake measured by FDG-PET 1 and 8 days after dose of ATV:ISO or ATV:TREM2 for 5×FAD;hTREM2 tg;TfRmu/hu mice (e) and WT;hTREM2 tg;TfRmu/hu mice (f) Scatter plot of individual FDG (SUV) values Dotted lines represent linear associations between interval after antibody dosing and FDG-PET quantification per group with a 95% confidence interval (n = 6 mice per group; two-tailed unpaired t-test for each timepoint) Regional correlation of biomarker alterations (5×FAD;hTREM2 tg;TfRmu/hu versus WT;hTREM2 tg;TfRmu/hu mice) between FBB-PET at 5 months and TSPO-PET (SUVH) (g) and FDG-PET (SUV) (h) at the group level These results suggest that ATV:TREM2 could rescue deficits in brain glucose metabolism potentially ameliorating this metabolic deficit found in AD we describe the mechanisms of action and cellular functions of ATV-enabled anti-TREM2 biologics including mouse-specific ATV:4D9 and human-specific ATV:TREM2 Our findings demonstrate that ATV enables improved biodistribution and activity of TREM2 antibodies The functional genetics demonstrate that LOF TREM2 LOAD risk variants disable protective microglial responses promoting TREM2 to improve microglia functions is a compelling therapeutic approach ATV not only improved brain uptake and biodistribution of our TREM2 antibody; it also enhanced TREM2 signaling at the cellular level through co-engagement of TREM2 and TfR We show that a key physiological effect of ATV:TREM2 is to improve the energetic capacity of microglia through enhanced fatty acid and glucose oxidation in mitochondria We also demonstrate that ATV:TREM2 increased microglial activity and glucose metabolism in an amyloid mouse model via TSPO-PET and FDG-PET imaging the impact of ATV on the cellular activity of a TREM2 antibody remained an open question We found that ATV altered TREM2 Fab function through two distinct mechanisms: (1) enhanced receptor clustering and (2) increased endosomal signaling Because ATV:TREM2 resulted in enhanced microglia responses compared to anti-TREM2 with equivalent brain IgG concentrations the molecular and cellular mechanisms identified in vitro are likely engaged in vivo These observations indicate that ATV:TREM2 has superior activity in addition to enhanced brain delivery and is a highly differentiated candidate biotherapeutic Our findings indicate microglia functions with disease-modifying potential based on the link to additional AD-relevant genes these data had not shown specific functional effects on microglia We show direct evidence of microglia proliferation induced by ATV:TREM2 which could help resolve pathology or serve as a renewal process to reset microglial state The proliferative response appears limited to a subset of microglia and factors regulating it are not yet understood ATV:TREM2 also improved microglial phagocytosis suggesting that ATV:TREM2 imparts multiple functions that could ameliorate disease indicating that microglial states are indeed tunable ATV:TREM2 increased FDG-PET in an amyloid mouse model thereby improving brain metabolism in a disease context Whether ATV:TREM2 induces glucose uptake cell autonomously in microglia or influences other CNS cell types remains an open question our results demonstrating improved brain exposure and biodistribution of a TREM2-activating antibody capable of enhancing microglial functions and improving brain metabolism suggest that ATV:TREM2 is a differentiated therapeutic candidate for the treatment of AD human TREM2 BAC tg mice and TfRmu/hu mice were crossed to generate 5×FAD;hTREM2 tg;TfRmu/hu (hemizygous;hemizygous;homozygous) and WT;hTREM2 tg;TfRmu/hu mice (non-carrier;hemizygous;homozygous) Mice are maintained on a C57BL/6J genetic background Mouse husbandry and experimental procedures were approved by the Denali Institutional Animal Care and Use Committee All animal experiments at the German Center for Neurodegenerative Diseases (DZNE) were performed in accordance with animal handling laws of the state of Bavaria (Germany) Housing conditions included standard pellet food and water provided ad libitum a 12-hour light/dark cycle at a temperature of 22 °C with maximal five mice per cage and cage replacement once per week with regular health monitoring iMG RNA was extracted with the RNeasy Plus Micro Kit (Qiagen 74034) to be used as input for bulk RNA-seq library generation using the QuantSeq 3′ mRNA-seq Library Prep Kit FWD for Illumina (Lexogen A01173) with the unique molecular identifier (UMI) second-strand synthesis add-on module Libraries were pooled and shipped to SeqMatic for sequencing on an Illumina sequencer scRNA-seq for WT mice was prepared with the Miltenyi Adult Brain Dissociation Kit (Miltenyi Biotec followed by enrichment with CD45 micro beads (Miltenyi Biotec was used for single-cell capture and library generation per the user guide Samples for the AD mouse model were prepared with the same Miltenyi dissociation kit but enrichment was performed via FACS sorting of live CD11b+ cells Samples were labeled with CellPlex reagents for multiplexing purposes and single-cell capture was performed using the Chromium Next GEM Single Cell 3′ Kit version 3.1 per the user guide Detailed information for bulk and scRNA-seq experimental procedures can be found in the Supplementary Methods section Pseudobulk differential expression analyses were carried out using limma/voom Detailed information about processing and analysis steps can be found in the Supplementary Methods; for analysis code right hemibrains were fixed by immersion in 4% paraformaldehyde (PFA) at 4 °C for 24 hours and then transferred to 30% sucrose solution for 2 days before sectioning coronally on a freezing microtome at a thickness of 40 μm Free-floating sections were permeabilized with 1× Tris-buffered saline solution containing 0.05% Tween (TBST) blocked with 5% donkey serum and incubated in primary antibodies overnight at 4 °C and a solution of secondary antibodies was then applied for 1 hour at room temperature Sections were washed in TBST before mounting and cover-slipping with ProLong Glass Antifade Mountant solution (Thermo Fisher Scientific Immunofluorescence was performed using the following primary antibodies: goat anti-Iba1 (Novus 1:500); guinea pig anti-Iba1 (Synpatic Systems 1:500); and goat anti-Axl (R&D Systems 1:25) and the following secondary antibodies: Alexa Fluor 488 donkey anti-Rabbit IgG (Jackson ImmunoResearch 1:500); Alexa Fluor 555 donkey anti-rabbit IgG (Thermo Fisher Scientific 1:500); Alexa Fluor 647 donkey anti-goat IgG (Thermo Fisher Scientific 1:500); and Alexa Fluor 647 donkey anti-guinea pig IgG (Jackson ImmunoResearch To quantify microglia coverage in the brains immunohistochemical staining by DAB precipitation coloring was also performed after goat anti-Iba1 primary incubation The sections with immunofluorescence staining were imaged and analyzed for microglia morphology See Supplementary Methods for details on microglia morphology quantification Antibody concentrations were quantified using a generic anti-human IgG sandwich-format ELISA Plates were coated overnight at 4 °C with donkey anti-human IgG (Jackson ImmunoResearch 709-006-098) at 1 µg ml−1 in sodium bicarbonate solution (Sigma-Aldrich Plates were washed 3× with PBS + 0.05% Tween 20 Assay standards and samples were diluted in PBS + 0.05% Tween 20 + 1% BSA (10 mg ml−1) A standard curve ranging from 0.41 ng ml−1 to 1,500 ng ml−1 (0.003–10 nM) was fitted using a four-parameter logistic regression Standards and diluted samples were incubated with agitation for 2 hours at room temperature HRP-conjugated goat anti-human IgG (Jackson ImmunoResearch was diluted in blocking buffer (PBS + 0.05% Tween 20 + 5% BSA (50 mg ml−1)) to a final concentration of 0.02 µg ml−1 and plates were incubated with agitation for 1 hour at room temperature plates were developed by adding TMB substrate for 5–10 minutes Animals were dosed with EdU solution (20 mg ml−1 sc-284628) via intraperitoneal (IP) injection at 80 mg kg−1 at day 0 (30 minutes after antibody treatment) animals were deeply anesthetized via IP injection of 2.5% Avertin The mice were then perfused intracardially with cold PBS Left hemibrain was snap-frozen on dry ice for biochemical analysis Right hemibrain was fixed in 4% PFA at 4 °C for 24 hours for sectioning and immunostaining For detecting EdU+ microglia in brain sections Click-iT EdU imaging kits (Thermo Fisher Scientific C10637) were used following the manufacturer’s instructions before the Iba1 immunofluorescence staining procedures Human TREM2/DAP12 is overexpressed in HEK293 cell line (RRID: CVCL_0045 ATCC: PTA-4488) by transfection of a dual promoter pBudCE4.1 vector driving expression of TREM2 under the CMV promoter and DAP12 the under the EF1a promoter Stable clones were isolated after zeocin selection (800 µg ml−1 for 10 days) and TREM2 was detected by flow cytometry (APC-conjugated rat anti-human/mouse-TREM2 monoclonal antibody The clone showing the highest TREM2 expression level was selected Phospho-Syk was measured by the AlphaLISA assay following the manufacturer’s protocol (PerkinElmer hTREM2-DAP12 HEK293 cells were plated at 50,000 cells per well the day before the assay Cells are stimulated with TREM2 antibodies for 5 minutes at 37 °C using Bravo liquid handler TREM2 antibody was mixed with a custom-made monoclonal TfR antibody that binds the ATV epitope in the apical domain (KD ~0.9 nM) at 37 °C for 30 minutes before adding to the cells Cells are lysed with lysis buffering containing 1 µM PMSF Data are collected from PerkinElmer EnVision plate reader hTREM2-DAP12 HEK293 cells were plated on 10-cm dishes coated with PDL 24 hours before antibody treatment Cell lysate was collected with 1-ml ice-cold IP buffer (Thermo Fisher Scientific 87787) containing 1× protease inhibitor (Cell Signaling Technology Cell lysate was normalized based on BCA measurement 200–400 µg of total lysate was used for each IP and was mixed with 2 µg of biotinylated capturing antibody at 4 °C overnight Normal IgG from the same species as the IP capturing antibody was used as binding control Antibody biotinylation was performed using an EZ-Link Sulfo-NHS-LC-Biotin Kit following the manufacturer’s instructions (Thermo Fisher Scientific AF1828) and normal goat IgG (R&D Systems a human monoclonal clonal antibody against TfR and an isotype IgG isotype antibody were made in-house for the study The immune complex was then precipitated using 20 µl of streptavidin-conjugated magnetic beads (Resyn Biosciences MR-STV005) and washed 4× with ice-cold 1× PBS buffer containing 0.05% Tween 20 25 µl of LDS containing 1× sample buffer (Thermo Fisher Scientific NP0007) and 1× reducing agent (Thermo Fisher Scientific NP0009) was added directly to the washed beads and incubated at 75 °C for 10 minutes Samples were then analyzed following standard western blot protocol The following antibodies were used for western detection: anti-TREM2 (R&D Systems 13-6800) and anti-actin (Cell Signaling Technology The pLVX-TRE3G-TREM2-BioID2 plasmid and the transponder pLVX-EF1a-Tet3G plasmid (Takara 631359) were introduced into the parental hTREM2-DAP12 HEK293 cells as stable cell line Cells were maintained in biotin-free media containing DMEM (Gibco 2–4 ng ml−1 of Dox was added to initiate TREM2-BioID fusion protein expression Cells were treated with antibody solution containing 100 nM TREM2 antibody and 2 µM biotin prepared in 1× PBS for 10 minutes at 37 °C Both no-Dox and no-biotin samples were included as negative control Cell lysates were prepared with RIPA buffer (Teknova R3792) containing 1× phosphatase/protease inhibitor (Cell Signaling Technology Normalized protein lysate containing 0.5–1 mg of protein was incubated with 20 µl of magnetic streptavidin beads (Resyn Biosciences Beads were then washed 4× with ice-cold RIPA buffer Captured protein was eluted with 2× NuPAGE LDS sample buffer (Thermo Fisher Scientific 20 mM biotin and 1× reducing agent (Thermo Fisher Scientific NP0009) and heated at 95 °C for 10 minutes AF1828) and anti-goat HRP secondary antibody (Thermo Fisher Scientific 81-1620) were used for detection with ECL reagent (Bio-Rad TREM2 biotinylation near 28 KD was normalized to the auto-biotinylation of TREM2-BioID band above 55 KD Human hTREM2-DAP12 HEK293 cells were plated at 35,000 cells per well 1 day before the experiment Cells were starved in serum-free DMEM for 1 hour before antibody stimulation at 10 nM and 37 °C for 10 minutes Transferrin is labeled with Alexa Fluor 647 transferrin conjugate (Invitrogen T23366) at 20 µg ml−1 during antibody stimulation Cells were fixed in 4% PFA for 10 minutes and permeabilized/blocked in PBS containing 0.3% Triton and 5% BSA Primary antibodies were diluted (1:250) in PBST buffer (0.06% Triton and 1% BSA in PBS) and used at 4 °C for 24 hours with anti-pSyk Tyr525/526 (Cell Signaling Secondary antibodies were diluted (1:500) in PBST buffer and used at room temperature for 45 minutes with Alexa Fluor 488 anti-human IgG (Jackson ImmunoResearch 109-545-003); Alexa Fluor 568 anti-rabbit (Thermo Fisher Scientific A10042); and Alexa Fluor 647 anti-mouse (Thermo Fisher Scientific Cells were washed with PBS 3× between each stain Data were acquired with Opera Phoenix High-Content Imager (PerkinElmer) at ×63 magnification Image quantification was performed using Harmony software (PerkinElmer version 5.1) with spot identification algorithm to quantify the fluorescence spot intensity by channel A masking algorithm was implemented in Harmony software to quantify the percent of IgG or pSyk spots co-localized within EEA1 or TfR Cell lysates were prepared in high-salt lysis buffer (Cell Signaling Technology 9803) with protease and phosphatase inhibitors (Cell Signaling Technology Normalized lysates were mixed with loading buffer (Thermo Fisher Scientific NP0007) and reducing agent (Thermo Fisher Scientific NP0004) and then resolved by electrophoresis using 4–12% NuPAGE (Thermo Fisher Scientific Semi-dry transfer was performed using the Trans-Blot Turbo System (Bio-Rad 1704150) with 0.2-µm PVDF transfer packs (Bio-Rad Transferred blots were blocked with Intercept Blocking Buffer (LI-COR 927-60001) and incubated with primary antibody overnight HRP-conjugated secondary antibodies were used for detection Washes were performed using TBST buffer containing 0.05% Tween 20 Blots were developed using ECL reagent (Bio-Rad 1705062) and imaged by the Li-COR Odyssey Fc imaging system Band signal intensity was quantified using Image Studio Lite software (LI-COR iMG were plated in cell carrier ultra 96-well plate (PerkinElmer 6055302) at a density of 25,000 cells per well and equilibrated for 72 hours in IMDM media (Thermo Fisher Scientific 20 ng ml−1 of granulocyte-macrophage colony-stimulating factor (GM-CSF) 20 ng ml−1 of IL-3 and 20 ng ml−1 of M-CSF Cells were dosed with 100 nM antibody prepared in reduced cytokine media containing 10% FBS (Cytiva Twenty nM mTOR kinase inhibitor AZD8055 (AZD and cells were treated for 96 hours before being fixed by 4% PFA at room temperature for 10 minutes and stained with EdU labeling kit (Thermo Fisher Scientific 62248) and cell mask (Thermo Fisher Scientific Fluorescent images were collected on Opera Phenix High-Content Imaging system equipped with a ×20 water lens (NA = 1.0) A total of nine fields per well were collected using three-step z-planes separated by 2 μm Total cell count was measured by counting DAPI+ nuclei using the ‘find nuclei’ function of Harmony software (PerkinElmer EdU+ nuclei were quantified from the 488 channel and frequency was calculated by normalizing to total DAPI+ nuclei CSF1R levels in brain homogenates and CSF were quantified using a commercial ELISA assay (Abcam and CSF was diluted 50-fold in NS diluent buffer supplied from the kit and the average reading was taken as the CSF1R protein level for that sample Mice were taken down 2 days after antibody dosing and brains were dissected after PBS perfusion and dissociated with the Adult Brain Dissociation Kit (Miltenyi Biotec Microglia number was measured by FACS using CountBright Absolute Counting Beads (Invitrogen C36950) and diluted to 500 microglia per µl in DPBS + 0.5% BSA The resulting cell suspension was mixed with pHrodo-green labeled myelin (50 µg ml−1 in DPBS + 0.5% BSA) or FAM-Aβ (200 nM in DPBS + 0.5% BSA) and incubated at 37 °C for 45 minutes with gentle mixing Cell suspensions were washed and stained with the following antibodies in FACS buffer (1% fatty acid-free BSA and 1 mM EDTA in PBS) for 25 minutes on ice: CD11b-BV421 (BioLegend 101251) and mouse Fc blocker (anti-mouse CD16/32 resuspended in FACS buffer with propidium iodide (Miltenyi Biotec strained through a 100-μm filter and then analyzed on a flow cytometer (BD FACSAria III) FCS files were then imported and analyzed in FlowJo software (version 10).The percentage of myelin+ microglia (pHrodo-green+ CD11b+) in the total CD11b+ microglial population was calculated See Supplementary Methods for details on myelin debris and Aβ fibril preparation and fluorescent labeling media was exchanged for media containing 100 nM ATV:TREM2 or ATV:ISO antibody and incubated for another 48 hours at 37 °C before imaging BODIPY staining or lipidomics extractions cells were incubated at 37 °C for 30 minutes in live cell imaging buffer (Life Technologies A14291DJ) containing 1 µM BODIPY 493/503 (Thermo Fisher Scientific D3922) and 1 drop per milliliter of NucBlue (Thermo Fisher Scientific Cells were fixed in 4% PFA and imaged using Alexa Fluor 488 and DAPI on an Opera Phenix High-Content confocal imager Lipid spots were analyzed using a spot-finding algorithm on Harmony software 100 µl of a 9:1 methanol:water solution containing 2 µl of internal standard mixture was added to the cells The plate was agitated on a shaker at 4 °C and 1,200 r.p.m for 20 minutes and centrifuged for 5 minutes at 300g A sample of supernatant was transferred to LC–MS vials and kept at −80 °C See Supplementary Methods for details LC–MS analysis iMG (20,000 cells per well) were seeded on a PDL-coated 96-well Agilent Seahorse XF Cell Culture microplate in media media replaced with substrate-limited media comprised of XF DMEM Antibodies were added to cells for a final concentration of 100 nM and incubated for 3 days and antibody was re-added to the washed cells to a final concentration of 100 nM Cells were imaged using bright-field microscopy to obtain cell counts for normalization Cells were then incubated for 1 hour in a non-CO2 incubator Ports on the sensor plate were filled according to the XF Long Chain Fatty Acid Oxidation Stress Kit or XF Glucose Oxidation Kit and cells were subjected to sequential injections of oligomycin (final concentration 1.5 µM) FCCP (2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile) (2 µM for fatty acid oxidation and 1.5 µM for glucose oxidation) and rotenone/antimycin A (0.5 µM each) etomoxir or UK5099 were added in port A at 4 µM or 3 µM Data were analyzed using the Agilent Seahorse Analytics online software to generate kinetic curves and calculate maximal respiration and spare capacity 5–6-month-old WT;hTREM2 tg;TfRmu/hu mice and 4.5-months-old 5×FAD;hTREM2 tg;TfRmu/hu mice (n = 6 each) were IP injected with ATV:TREM2 or an isotype control antibody at 14 mg kg−1 and 10 mg kg−1 Mice were subjected to either TSPO-PET or FDG-PET imaging 24 hours after dose Microglia activation and brain glucose metabolism were followed longitudinally with further PET scans on days 4 and 8 after antibody administration Male and female mice were distributed evenly among both antibody treatment and PET imaging groups See Supplementary Methods for details on PET imaging procedures and quantification All statistical analyses performed were two-tailed Data normality was examined by the Shapiro–Wilk test F-test or Brown–Forsythe test was used to assess data homoscedasticity Tukey’s multiple comparisons test or multiple t-test was used depending on data groups unpaired t-test with Welch’s correction test or Kruskal–Wallis test was used Data with normal distribution but unequal variance were assessed with Mann–Whitney test or Brown–Forsythe and Welch’s ANOVA test Statistical P values are shown on graphs denoted with ‘P = ’ Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The metabolomics data were uploaded to the MetaboLights repository with study ID MTBLS6543. Source data are provided with this paper All R and Python code used to produce transcriptomics analyses and imaging morphometric analyses are available through GitHub at the following URL: https://github.com/denalitherapeutics/Lengerich_natneuro_2022 A genome-wide association study with 1,126,563 individuals identifies new risk loci for Alzheimer’s disease Emerging microglia biology defines novel therapeutic approaches for Alzheimer’s disease TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model TREM2—a key player in microglial biology and Alzheimer disease ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157 Sequential proteolytic processing of the triggering receptor expressed on myeloid cells-2 (TREM2) protein by ectodomain shedding and γ-secretase-dependent intramembranous cleavage TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2 TREM2 deficiency impairs chemotaxis and microglial responses to neuronal injury TREM2 regulates microglial cholesterol metabolism upon chronic phagocytic challenge A unique microglia type associated with restricting development of Alzheimer’s disease Trem2 deletion reduces late-stage amyloid plaque accumulation and exacerbates axonal dystrophy and dendritic spine loss in the PS2APP Alzheimer’s mouse model Diet-dependent regulation of TGFβ impairs reparative innate immune responses after demyelination Oxidized phosphatidylcholines found in multiple sclerosis lesions mediate neurodegeneration and are neutralized by microglia Variant of TREM2 associated with the risk of Alzheimer’s disease Genetic meta-analysis of diagnosed Alzheimer’s disease identifies new risk loci and implicates Aβ Alzheimer’s disease-associated TREM2 variants exhibit either decreased or increased ligand-dependent activation Whole exome sequencing study identifies novel rare and common Alzheimer’s-associated variants involved in immune response and transcriptional regulation An Alzheimer-associated TREM2 variant occurs at the ADAM cleavage site and affects shedding and phagocytic function TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer’s disease-associated H157Y variant and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions Alzheimer’s-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia Increased soluble TREM2 in cerebrospinal fluid is associated with reduced cognitive and clinical decline in Alzheimer’s disease Soluble TREM2 in CSF and its association with other biomarkers and cognition in autosomal-dominant Alzheimer’s disease: a longitudinal observational study TREM2-activating antibodies abrogate the negative pleiotropic effects of the Alzheimer’s disease variant Trem2R47H on murine myeloid cell function TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis Therapeutic Trem2 activation ameliorates amyloid-beta deposition and improves cognition in the 5×FAD model of amyloid deposition Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region Anti-human TREM2 induces microglia proliferation and reduces pathology in an Alzheimer’s disease model Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer’s disease Engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer’s disease models Brain delivery of therapeutic proteins using an Fc fragment blood–brain barrier transport vehicle in mice and monkeys Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic Molecular architecture determines brain delivery of a transferrin receptor-targeted lysosomal enzyme Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia Temporal tracking of microglia activation in neurodegeneration at single-cell resolution Microglia in Alzheimer’s disease at single-cell level Are there common patterns in humans and mice Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies An improved smaller biotin ligase for BioID proximity labeling iPSC-derived human microglia-like cells to study neurological diseases Colony-stimulating factor 1 receptor signaling is necessary for microglia viability unmasking a microglia progenitor cell in the adult brain AD-linked R47H-TREM2 mutation induces disease-enhancing microglial states via AKT hyperactivation In-vivo evaluation of indium-111-diethylenetriaminepentaacetic acid-labelling for determining the sites and rates of protein catabolism in mice TREM2 regulates microglial cell activation in response to demyelination in vivo Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain Lipid and lipoprotein metabolism in microglia Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure Mitochondrial dynamics at the interface of immune cell metabolism and function and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation Microglial activation states drive glucose uptake and FDG-PET alterations in neurodegenerative diseases The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism Tracking pathophysiological processes in Alzheimer’s disease: an updated hypothetical model of dynamic biomarkers FDG-PET as an independent biomarker for Alzheimer’s biological diagnosis: a longitudinal study TREM2 ameliorates neuroinflammatory response and cognitive impairment via PI3K/AKT/FoxO3a signaling pathway in Alzheimer’s disease mice Brain delivery and activity of a lysosomal enzyme using a blood–brain barrier transport vehicle in mice Loss of TREM2 rescues hyperactivation of microglia but not lysosomal deficits and neurotoxicity in models of progranulin deficiency A locked immunometabolic switch underlies TREM2 R47H loss of function in human iPSC-derived microglia TREM2 maintains microglial metabolic fitness in Alzheimer’s disease limma powers differential expression analyses for RNA-sequencing and microarray studies Korotkevich, G. et al. Fast gene set enrichment analysis. Preprint at https://www.biorxiv.org/content/10.1101/060012v3 (2021) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles The Molecular Signatures Database (MSigDB) hallmark gene set collection Integrated analysis of multimodal single-cell data Efficient proximity labeling in living cells and organisms with TurboID Age-related myelin degradation burdens the clearance function of microglia during aging Automated spatial brain normalization and hindbrain white matter reference tissue give improved [18F]-florbetaben PET quantitation in Alzheimer’s model mice Glial activation and glucose metabolism in a transgenic amyloid mouse model: a triple-tracer PET study Download references Rakhit for their work at the early stage of the project Sandmann for input on RNA sequencing data access Raju for cloning and optimization of the BioID in vitro system These authors contributed equally: Bettina van Lengerich German Center for Neurodegenerative Diseases (DZNE) Munich Cluster for Systems Neurology (SyNergy) conceived of the study idea and approaches are full-time employees and/or shareholders of Denali Therapeutics participated on one advisory board meeting of Biogen and received a speaker honorarium from Novartis and Roche is chief advisor of ISAR Bioscience and a member of the advisory board of AviadoBio is a full-time employee of Takeda Pharmaceuticals a clinical development partner of Denali Therapeutics Nature Neuroscience thanks Takanori Yokota and the other reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations (a) UMAP projections of individually processed data sets for WT; TfRmu/hu timecourse and TfRmu/hu; AppSAA studies Microglia are color coded according to their experimental group (b) Combined UMAP of integrated data by study Microglia are color coded by unbiased cluster assignment (c) Stacked barplots showing the proportion of microglia per biological replicate by cluster and each bar represents a biological replicate within that group Barplot color scheme is consistent with clusters in b (d) Feature plots showing expression of selected individual genes Microglia are color coded according to log normalized expression of each gene (e) Antibody concentrations detected in whole brain lysate from either WT; TfRmu/hu or TfRmu/hu; AppSAA mice dosed with 10 mg kg−1 ATV:ISO or ATV:4D9 (n = 8 mice/group except for ATV:4D9 WT;TfRmu/hu and 4D9 APPSAA; TfRmu/hu n = 4 mice/group) (a) Representative morphometric images of microglia in the cortex 1 day post dose with ATV:ISO or ATV:4D9 (10 mg kg−1) at day 1- (c) Percentage of microglia in the responsive cluster as a proportion of all segmented microglia over time (n = 3 male mice per group two-tailed unpaired t-test between ATV:ISO and ATV:4D9 at day 1 (d) Volcano plot showing the top 6 differentially high and low normalized features comparing the responsive cluster to the homeostatic cluster (e) Heatmap of normalized features for all segmented microglia (1,143 total cells) over time measured across 65 morphometric and immunohistochemical features grouped by treatment (rows) with features hierarchically clustered (columns) (f) Representative images of cortical brain sections co-stained with Iba1 and CD74 at 1- 28-days days post 10 mg kg−1 dose of ATV:ISO or ATV:4D9 CD74+ microglia are noted with white arrows (g) Mean intensity of CD74 staining within segmented IBA1+ microglia normalized to background CD74 intensity at each timepoint (n = 5 mice/group (h) Representative images of cortical brain sections stained for IBA1 and AXL 1 day post dose of ATV:ISO or ATV:4D9 Double positive microglia are noted with white arrows (i) Quantification of mean intensity of AXL staining within segmented IBA1+ microglia normalized to background (n = 5 mice/group The binding epitope of ATV:4D9 antibody is located 12-amino acids N-terminal of the ADAM cleavage site at His157 (c) FACS analysis of cell binding of ATV:TREM2 to hTREM2-DAP12 HEK293 or parental cells Endogenous TfR expression on HEK293 cells drives weak binding observed for ATV:ISO and ATV:TREM2 (n = 3 independent experiment (d) FACS detection of ATV:TREM2 (100 nM) binding to WT and TREM2 KO iMG with isotype control (ATV:ISO) (e) Soluble TREM2 measured in the supernatant of hTREM2-DAP12 HEK293 cells dosed with ATV:TREM2 for 24 h shows dose-dependent reduction of sTREM2 to levels similar to 1 uM GM6001 (n = 3 independent experiment (f) ATV:TREM2 and lipid ligands induce pSyk signaling in iMG 24 h post antibody exposure (n = 3 independent experiments (g) Human monocytes cultured in limited M-CSF with plate coated ATV:TREM2 or ATV:ISO shows dose-dependent activity of ATV:TREM2 (EC50 0.95 +/− 0.45 nM) Representative data from one out of four human donors are shown (a) Representative Western blot of co-IP of TREM2 with TfR hTREM2-DAP12 HEK293 cells were treated with 100 nM ATV:TREM2 (b) Co-IP quantification of Western blot from (A) (n = 6 independent experiments; two-tailed paired t-test for ISO vs anti-TREM2; two tailed Wilcoxon test ATV:ISO vs ATV:TREM2 (c) Schematic illustration of cis- and trans-activation models that could mediate pSyk enhancement by ATV:TREM2 (d) Western blot validation of TfR knockdown in the TfRRNAi cell line (e) Cell based cis/trans assay indicates ATV:TREM2 enhances pSyk activity in cis Relative pSYK signal is expressed as raw pSYK AlphaLisa value normalized to ATV:TREM2 treated control (n = 3 independent experiment (f) Normalized pSyk signal measured by AlphaLisa assay TfRRNAi cells were treated with 10 nM anti-TREM2 or ATV:TREM2 pre-incubated with a dose titration of recombinant TfR protein for 5 min at 37 °C (n = 3 independent experiment (g) Normalized pSyk signal detected by AlphaLisa TfRRNAi cells were treated with 10 nM anti-TREM2 or ATV:TREM2 pre-incubated with a dose titration of a secondary anti-human IgG Fc antibody for 5 min at 37 °C (n = 3 independent experiment mean ± SEM) (h) Immunofluorescence microscopy of hTREM2-DAP12 HEK293 cells demonstrates reduction of surface TREM2 levels with ATV:TREM2 vs anti-TREM2 consistent with re-distribution of the receptor from the plasma membrane to endosomes (n = 4 independent experiments except for anti-TREM2 MV (n = 3) (i) hTREM2-DAP12 HEK293 cells dosed with antibody for 10 minutes shows that at similar amounts of bound antibody detected by anti-IgG (representing 5 nM of ATV:TREM2 and 10 nM of anti-TREM2 mean ± SEM(j) Images depicting masking algorithm used to identify whether TfR-Alexa-647 labeled recycling endosomes (rainbow spots in middle images) either contain (green spots in right-most images) or do not contain (red spots in right-most images) IgG spots (white spots in left-most image) upon dosing with anti-TREM2 (top row) or ATV:TREM2 (bottom row) for 10 minutes with 10 nM antibody (k) Representative images for hTREM2-DAP HEK293 cells dosed with 10 nM antibody for 10 minutes including 20 ug mL−1 TfR-Alexa-647 IF shows ATV increased colocalization of antibody with pSyk in early endosomes (l) Quantification of percent of IgG or pSyk spots localized within Tf-positive endosomes (n = 3 independent experiments (a) Representative Western blot images of phosphorylation of 4EBP1 (T37/46) and ERK1/2 (T202/Y204) in WT iMGs treated for 96 h with 100 nM ATV:TREM2 or an isotype control (b) Quantification of p4EBP1 (T37/46) and pERK1/2 (T202/Y204) normalized to actin Relative expression was calculated by normalizing to PBS vehicle control for each experiment (n = 4 independent experiments (c) Representative Western blot images of total protein levels of mTOR and AKT in WT iMG treated for 96 h with 100 nM ATV:TREM2 or an isotype control (d) Quantification of total mTOR and AKT protein normalized to actin Relative expression was calculated by normalizing to PBS control for each experiment (n = 4 independent experiments e) Representative Western blot images for p-mTOR (S2448) p4EBP1 (T37/46) and pERK1/2 (T202/Y204) showing inhibition of mTOR pathway activation in WT iMG co-treated with 20 nM AZD8055 and 100 nM ATV:TREM2 after 96 h (f) Quantification of phosphorylation targets shown in (E) Phosphorylation signals were normalized to actin (g) Heatmap of human cytokine profiling in supernatant from WT iMG treated with 100 nM ATV:TREM2 for 96 h Media collected from iMG treated with 10 ng mL−1 LPS for 24 h was used for comparison (a) Antibody levels detected in brain lysates by ELISA shows increased brain exposure of ATV:TREM2 (30 mg kg−1) compared anti-TREM2 (30 mg kg−1) and comparable brain exposure for ATV:TREM2 at 10 mg kg-1 and anti-TREM2 at 30 mg kg−1 at day 4 post-dose (n = 5 mice/group except for ATV:TREM2 3 mg kg−1 (n = 4)) (b) ATV:TREM2 increases CSF1R in the brain compared to brain exposure matched anti-TREM2 day 2 post dose (n = [8 mean ± SEM) (b-c) Circles represents male mice and triangle represents female mice (d) Detection of antibody concentrations show matched brain exposure of three ATV:TREM2 molecules with different ATV affinities 1-day post-dose (n = 5 mice/group) (e-f) High and mid-affinity ATV:TREM2 molecules induce comparable increase of CSF1R in the brain (e) n = 5 mice/group except for Vehicle (Veh) and day 4 10 mg kg−1 8,000 nM (n = 4 compared to vehicle group) and CSF (f) (n = 5 mice/group except for Veh and day 4 5 mg kg−1 110 nM n = 4) Dunnett’s multiple comparisons test for day 1 Kruskal-Wallis test for day 4) day 1 and 4 post dose whereas low-affinity ATV:TREM2 induces a weak elevation of CSF1R in CSF at day 4 (n = 4-5 mice/group (g) Schematic of experimental approach to evaluate in vivo dosed antibody impact to microglial phagocytosis ex vivo or ATV:ISO was administrated to human TREM2 tg; TfRmu/hu KI mice and brain microglia were isolated 2 days post dose for ex vivo myelin phagocytosis analysis The same method was used to assess amyloid phagocytosis (h) FACS gating strategy to quantify pHrodo-myelin positive microglia After treatment with pHrodo-green myelin and staining cell suspensions containing microglia were analyzed using BD FACS Aria III Single cells were separated from debris by FSC and SSC characteristics Live microglia were identified as a population of CD11b+ and propidium iodidenegative cells pHrodo-myelin uptake was then quantified in 20,000 microglia recorded from each sample (i) Antibody concentrations were detected in brain lysates by ELISA which shows comparable levels of ATV:TREM2 and anti-TREM2 day 2 post dose (n = [9 (j) ATV:TREM2 induces transcription of genes associated with phagocytosis Lgals3 mRNA levels were detected in isolated brain microglia after peripheral administration of 10 mg kg−1 ATV:TREM2 1- Graphs represent bulk mRNA measured by RNA-seq Data shown as log2 counts per million (with a pseudocount of 1 added) in each biological replicate (n = 8 mice/group Dunnett’s multiple comparisons for Itgax and Lgals3 (a) Representative autoradiography images of sagittal brain sections from WT; hTREM2 tg; TfRmu/hu mice 96 h after administration of ATV:TREM2 and ATV:ISO radiolabeled with [111In]DOTA or [125I]SIB (b,c) Longitudinal SPECT/CT imaging quantification of whole-brain uptake of single dose (100 µCi radiolabeled with [125I]SIB (b) or [111In]DOTA (c) in WT; hTREM2 tg; TfRmu/hu mice Whole brain %ID/g was corrected for contribution from cerebral blood volume Data are represented as mean +/− SEM (n = 3 mice/group) (d) Percent of catabolized ATV:TREM2 in several brain regions 96 h after single dose exceeds that of ATV:ISO control [111In]DOTA and [125I]SIB signal was quantified by ex vivo gamma counting in resected brain regions and percent of catabolized antibody was estimated by subtracting %ID/g for [125I]SIB signal from that of [111In]DOTA signal (n = 4 mice/group one-way ANOVA with Dunnett’s multiple comparison test for each region except for cerebellum which applied Brown-Forsythe and Welch ANOVA tests (a) Additional species of triglycerides (TG) and short chain carnitines modulated in iMG with ATV:TREM2 treatment (n = 3-5 independent experiment (b) ATV:TREM2 does not modulate TG in PLCG2 KO iMG (n = 3 independent experiment (c) Maximal respiration measured by Seahorse fatty acid oxidation kit is reduced in both TREM2 KO and PLCG2 KO iMG (n = 3-4 independent experiment (d) ATV:TREM2 increased spare capacity measured by Seahorse fatty acid oxidation kit (n = 8 independent experiment (e) ATV:TREM2 does not modulate maximal respiration or spare capacity in TREM2 KO iMG (n = 4 independent experiment (f) ATV:TREM2 does not modulate maximal respiration and spare capacity PLCG2 KO iMG (n = 4 independent experiment (g) RNAseq analysis of brain microglia isolated from hTREM2 tg; TfRmu/hu mice dosed with 10 mg kg−1 ATV:TREM2 GSEA for top pathways based on a p-value cutoff of 0.05 for up- or downregulated gene sets 1 day post ATV:TREM2 dose Supplementary Tables 1–3; Extended Data Figs 1–4; Supplementary Materials and Methods; and References Download citation DOI: https://doi.org/10.1038/s41593-022-01240-0 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Journal of Neuroimmune Pharmacology (2025) Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Today an opening ceremony was held for a new cobalt mine near Salmon which represents a significant day for the domestic supply chain of the metal Australian mining company Jervois Global Limited officially opened operations today but the mine won't be operating at full capacity until the first quarter of 2023 Executive General Manager of Idaho Cobalt Operations we will produce about 2000 tons of cobalt in concentrate each year So that's enough for roughly 400,000 electric vehicles," Lengerich said Forest Service land about 40 miles west of Salmon and is on track to be the only primary cobalt operation in the country The "Idaho Cobalt Belt" represents an opportunity for a more secure way for American companies to get their hands on the critical material needed for various industries But current supply of the mineral used in electric vehicles and defense applications comes primarily from Africa and is processed and refined in China The operations abroad have faced criticism due to human rights violations UNICEF estimates thousands of children are working in artisanal mines located in the Democratic Republic of Congo The cobalt concentrate produced at the Jervois mine in Idaho will be shipped to Brazil for refinement our only option is to take our cobalt concentrate overseas." Governor Brad Little attended the opening ceremony and toured the facility Thanks for visiting Thanks for visiting Tom Saint-Juvin in the final stretch runs passed his competitor to take the GOLD!?? pic.twitter.com/dD629T9aaZ Thanks for visiting First broken record of the GLVC Championships! Lengerich broke the school and GLVC record in the hep! pic.twitter.com/8F0g6snenQ Hurt passes his coaches record! Cole Hurt broke Coach Trace Oswalt's record in the decathlon with 6476 points! pic.twitter.com/d05PM1qwbV Field takes her teammates record! Fields broke Lengerich's long jump record with 5.86m! pic.twitter.com/4unuJWcqtD Thanks for visiting This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Alliance Defending Freedom sent two complaints on Wednesday to the Office for Civil Rights (OCR) at the U.S asking the agency to investigate unlawful policies harming students and female athletes In its first complaint, ADF attorneys are asking OCR to investigate Washington state’s athletic policy which permits males to compete in women’s sports two female high school track and field athletes – Kora Lengerich a freshman at Gonzaga Preparatory School in Spokane a senior at Prosser High School in Prosser Washington – are being forced to compete against a male athlete The male – who previously competed in the high school boys’ category – now runs on the female track team at East Valley High School in Spokane and “dominates the girls’ 400-meter race,” ADF notes he won the state championship for the girls’ 400 meter and is likely to do so again this year Lengerich has already lost a podium spot to the male athlete and Hoefer will likely have to compete against him soon The girls are “urging the OCR to step in and prevent this violation of Title IX and protect the right of women and girls to compete fairly and safely by preserving a women’s-only sports category.” “It is fundamentally unfair that, despite all of their hard work and discipline, Soliel and Kora may never get to know the thrill of victory,” said ADF Legal Counsel Suzanne Beecher in a statement “because Washington forces them to race against males with inherent biological advantages.” She added In its second complaint ADF joined the Wisconsin Institute for Law and Liberty and Parents Defending Education urging the DOE to investigate Milwaukee Public School’s secret “gender transition” policy The policy permits school staff to create “Gender Support Plans” and “Gender Communication Plans” for minor students without notifying their parents and then omit those plans from the students’ records to keep them secret The complaint alleges the policy violates federal law – including the Family Education Rights and Privacy Act and the Protection of Pupil Rights Amendment “Parents, not the government, have the right to direct the upbringing, education, and health care of their children,” said ADF Senior Counsel Kate Anderson, director of the ADF Center for Parental Rights, in a statement Under the Biden administration, the DOE amended Title IX rewriting “sex” to include “gender identity” in an attempt to force schools nationwide to permit males into female sports President Trump’s executive order protecting women’s sports, signed in February, directs the secretary of education to “take all appropriate action to affirmatively protect all-female athletic opportunities.” It also directs the secretary to “prioritize enforcement against schools that violate the policy.” The DOE has been tasked with defending girls and women’s sports ensuring young women are protected from males who would do them harm The Daily Citizen is hopeful the DOE will quickly step in and restore common sense and biological reality in both cases above If you’re concerned about what your child is being taught in school, check out this updated, free resource from Focus on the Family and Family Policy Alliance: Equipping Parents for Back-to-School We want parents to feel confident and equipped to manage issues affecting public – and private and online – schooling. The FREE downloadable resource helps you be aware of what’s going on in your child’s classroom and offers guidance for how to advocate for your child in the school year ahead Counseling Consultation & Referrals Transgender Resources Addressing Gender Identity with Honesty and Compassion The Journey Back to My True Identity What is ‘Gender Identity’? Payton McNabb, Injured Volleyball Player, Wins Title IX Victory Department of Education Launches Multiple Investigations Into Title IX Violations Don’t Let the Media Deceive You About Trump’s Order Protecting Female Athletes Trump Signs Executive Order Protecting Women’s Sports and Spaces Video: Seven-Year-Old's Confidence Soars After Ordering Chick-Fil-A By Himself Radical Colorado ‘Transgender’ Bill Threatens Parents’ Rights and Free Speech Zachary Mettler Zachary Mettler is a writer/analyst for the Daily Citizen at Focus on the Family Mettler earned his Bachelor’s degree from William Jessup University and is an alumnus of the Young Leaders Program at The Heritage Foundation his written pieces have appeared in the Daily Wire President Trump Announces New Commission to Defend Religious Liberty Focus on the Family Testifies Against Nightmare Bill Colorado’s Radical ‘Trans’ Legislation Advances Florida AG Launches Office of Parental Rights Tennessee Bill Guards Conscience Protections for Healthcare Providers to the late Herman and Amelia (Ulman) Lengerich Linda (Kevin) Wellman of Decatur; 41 grandchildren; 43 great grandchildren; her siblings Joe (Jane) Lengerich of Decatur; sisters-in-law Marjorie and Jean Lengerich; five brothers Alfrieda Welling OP; Hilbert (Donna) Welling Mary Ellen was a 1947 graduate of Decatur Catholic High School She was a member of Holy Trinity Catholic Church the Catholic Ladies of Columbia and taught religious education for many years She was also a member of the Bryant Senior Citizens Mary Ellen enjoyed flower gardening and was known for decorating cakes for many weddings and other occasions Family and friends were very important to her and she loved spending time with them A Mass of Christian Burial will be 10:30 am Tuesday Calling is 2:00 pm-8:00 pm Monday and 9:00 am-9:45 am Tuesday at Brockman - Boeckman Funeral Home Condolences may be directed to www.brockmanboeckmanfh.com ?? | Congrats to @UIndyXCTF's Sabrina Robison, the 2?0?2?3? #GLVCtrack pole vault champion! A new program record! ?? pic.twitter.com/8ko1EfjWKV ?? | Congrats to @UIndyXCTF's Ellie Lengerich, the 2?0?2?3? #GLVCtrack pentathlon champion! A new program record!?? pic.twitter.com/m6utoIYALN ?? | Congrats to @UIndyXCTF's Treyton Arnold, the 2?0?2?3? #GLVCtrack pole vault champion! A new program record! ?? pic.twitter.com/WvRSanT1VQ Thanks for visiting the "Gem State" has plenty of metals underground — and that is what's drawing global markets to the area Cobalt mining resumed in Idaho for the first time in decades marked by an opening ceremony for a mine operated by Australian company Jervois Global Ltd this relationship is going to be something that's going to help Idaho for a long who attended the ceremony and addressed the crowd "It's going to make America and Australia.. have a better relationship and provide for the future of this country renewable energy that's going to sustain this country and the world into the future," Little continued An expanding green technology market combined with international supply chain challenges has highlighted the need and opportunity for domestic production of rare metal elements like cobalt Cobalt is a necessary component for electric vehicle batteries begin feeding ore into our mill and the production of (cobalt) concentrates," Matt Lengerich executive general manager for Jervois' Idaho Cobalt Operations "The security of our supply chains and our access to critical minerals is important for us as U.S American industry is at the whim of foreign supply chains when it comes to certain materials needed for clean-energy technologies many public officials want to see a stronger domestic supply of rare-earth elements like cobalt and lithium “We expect to produce enough cobalt for the batteries of 2.8 million electric vehicles," Lengerich said of the mine's overall economic contribution Right now, the majority of these materials are mined and refined abroad. According to the U.S. Geological Survey the Democratic Republic of Congo supplies the majority of the world's mined cobalt while China supplies the majority of the world's refined cobalt In August, the Biden Administration launched a $675 million dollar infrastructure program to expand and support these supply chains The White House estimates global demand for critical materials is expected to increase 400-600% in the decades to come shifting global supply chains will take time Jervois' cobalt mine 40 miles west of Salmon is an early step “I think it's really important to recognize that Australia and America have a long history of collaborating together," Lengerich said Lengerich said the company estimates 2000 tons of cobalt concentrate will be produced annually at the Idaho location it will have to be shipped to Jervois' cobalt refinery located in Brazil afterward so at the moment our only option is to take our cobalt concentrate overseas," Lengerich said It isn't clear on how much refined cobalt will return back to the country afterwards what the markets look like and where the demand for cobalt is but that is certainly one possibility is that that metal would all come back into the U.S.," Lengerich explained The mine's lifespan is currently seven years long but Lengerich said there could be more cobalt deposits available and that ultimately the mine could be in operation longer Idaho environmental watchdogs are keeping track of an increase of mining proposals in the state "A lot of new technologies that are emerging that are utilizing a lot of the minerals that we happen to have here," said Nick Kunath a conservation associate Idaho Rivers United (IRU) IRU is an organization focused on river health and conservation across the state. Recently, the group successfully advocated for drill sites to be removed and moved at the Colson-Cobalt Copper 3 exploratory mining location just north of the confluence of the Middle Fork and Main Salmon River "We are trying to keep track of where these proposals are taking place," Kunath said “It's not necessarily the mineral itself that's of concern but it's more of just the impact that a large-scale mine can have on the ecosystem." Kunath said if a mine is in close proximity to a watershed "If there's a mine or a project that has potential to have some negative impacts on water quality or fisheries So there's things that we kind of get a little bit more engaged upon and stay involved with," Kunath said external relations director for Idaho Conservation League (ICL) echoed the concern over proximity to water "It is the number one toxic polluter in the United States and so ensuring that that any kind of waste or discharge from a mine is properly treated." ICL has tracked mining operations in the state Oppenheimer said mining can result in heavy metal contamination so ICL works with the government and private companies like Jervois to safeguard the environment "If it was habitat for endangered species or wildlife before the mining project we want to make sure that that habitat is available after the mining project concludes," Oppenheimer explained ICL is working with Jervois in an additional vein to fund conservation work with the Upper Salmon Conservation Action Program "(It) really tries to put some dollars on the ground even before they started digging any holes or started removing any minerals," Oppenheimer said The joint funding from Jervois mining is already flowing to projects involving wildlife habitat and water quality "If Idaho can be part of a clean energy revolution we think that we should be part of that discussion," he said The Jervois site near Salmon has another additional environmental safeguard within their operations: a water treatment plan According to the sites' General Manager Matt Lengerich it was the first facility commissioned and built on location "That water treatment plant actually is completely a backstop We don't expect to have to treat water during the life of the mine," Lengerich said "But that water treatment plant exists in the event that we do need to treat water we have that ability and we can treat it before it's discharged." The site is expected to be operating at full capacity by Quarter 1 of 2023 With the departure of his number one assistant trainer Ann-Christin Wienkamp German professional dressage trainer Oliver Oelrich has tightened the co-operation with his student Florine Kienbaum who has now started to compete some of his horses The 27-year old Kienbaum has been a member of the German team as a youth rider She competed at the 2008 European Pony Championships on Going East at the 2010 European Junior Riders Championships on Good Morning M and with Don Windsor she won numerous team medals and kur bronze at the 2012 and 2014 European Young Riders Championships In 2015 Kienbaum made the transition to Under 25 Grand Prix level with Doktor Schiwago and won double individual silver at the inaugural European Under 25 Championships in Hagen in 2016 In 2018 she made the transition to the senior division after choosing a career in horses as a professional rider For the past 10 years Kienbaum has been training with Oliver Oelrich and has relocated her own business out of his yard in Lengerich With the departure of Wienkamp as Oelrich's number one show rider "Since last year I have my own horses also in Lengerich and so it has offered itself to intensify our cooperation," Kienbaum told Eurodressage but Olli and I support each other in the training of young talents With local competitions starting up at great speed in central and northern Germany after corona restrictions led to the complete standstill of the show circuit for two months Kienbaum made her competition debut on some of Oelrich's entrusted horses at the CDN Nottuln on 23 - 24 May 2020 She won a developing Prix St Georges horse test on the 8-year old Oldenburg Bacanto (by Bordeaux x Don Romantic) and rode Andreas Helgstrand's 8-year old Oldenburg mare Florine OLD (by Foundation x Lauries Crusador xx) to a 9th place The chestnut mare was previously shown by Maike Mende and named second reserve for the 2019 World young horse championships She sold to Helgstrand but is kept in Germany with Oelrich to be developed and sold She further saddled the 7-year old KWPN gelding Inspire (by Johnson x Painted Black) Related LinksAnn-Christin Wienkamp Goes Independent, Relocates to LienenDeutsche Bank Equestrian Sport Academy Announces 2019 Grant Recipients Stalls for Rent at Durondeau Dressage in Peer, Belgium Exceptionally Well Located Equestrian Facility in Wellington, Florida Well-built Equestrian Estate With Multiple Business Opportunities in Sweden Stable Units for Rent at Lotje Schoots' Equestrian Center in Houten (NED) For Rent: Several Apartments and Stable Wing at High-End Equestrian Facility Stable Wing Available at Reiterhof Wensing on Dutch/German border Real Estate: Well-Appointed Country House with Extensive Equestrian Facility in the U.K. Rémi Blot we will replace an existing wind turbine at the Lengerich wind farm with a new As well as supplying four times more households with green electricity this brand new turbine will allow us to test the erection of a wind turbine on a prefabricated foundation for the first time This saves time and also reduces the CO2 footprint of our wind turbine once again.” RWE has decided on an innovative repowering project for a wind turbine in Emsland that sets an example in the field of sustainability which has been in operation at the Lengerich wind farm since 2003 is being replaced by a modern 5.7 megawatt turbine This will enable the significantly more powerful wind turbine to supply around 4,000 households with climate-neutral electricity in the future the existing turbine is producing enough green electricity to meet the needs of 1,000 households But the new plant will have a positive impact on the environment for another reason RWE will use a prefabricated foundation for the construction of the new plant The foundation developed by Smart & Green Anker Foundations consists of 100 percent precast elements produced in the concrete plant Only a third of the amount of steel and concrete normally used for poured standard foundations is used Since all parts can be produced in advance in a concrete plant the construction time is also reduced considerably as construction can take place in almost any weather less expensive and more environmentally friendly: instead of using 120 concrete mixers the parts are delivered by around 30 lorry trips and then bolted together on site They can be easily dismantled again in case of later deconstruction The certification process for the innovative foundation is currently underway This will be used to apply for a modification permit for the wind turbine that has already been approved The dismantling of the old wind turbine is planned for spring of next year which is scheduled to start operation in the fourth quarter of 2023 The hub height is 118 metres and the foundation will weigh around 800 tonnes.  he wanted to honor her with a song at the funeral the parish priest denied Hakes’ request to sing adding more pain to an already painful time Hakes’ family are longtime parishioners at St. Mary of the Assumption Church in Decatur, Indiana. Generations of the family, including his grandmother, were part of the community there, and Hakes had even sung at the church before, reported WANE banned Hakes from singing at the parish until the “present situation” was resolved in the letter explain what the “present situation” is  One of the issues mentioned in the letter that would ban people from liturgical roles was “openly participating in unchaste same-sex relationships.” Father Lengerich made his thoughts known in a letter to the grieving grandson The letter also said that scandal is caused by someone “openly advocating” for same-gender relationships He claimed there were “several LGTB parishioners who have openly declared their intentions to embrace a homosexual lifestyle” and therefore do no receive communion at Mass nor serve in any parish liturgical ministries The priest told Hakes that he could sing to honor his grandmother “as long as it is outside of the Mass and outside of the Church,” even suggesting the post-burial luncheon as a possible moment He concluded the letter saying the parish did want Hakes present and did “want to enter into a real dialogue and conversation.” Hakes claimed that Fr Lengerich based his claims about the gay man’s sexual life on a picture posted to Facebook several years ago of Hakes celebrating Pride The grandson told WANE that Lengerich “had judged me and really formed an opinion about me without ever communicating with me .All of a sudden I felt very ostracized” from the parish that had always welcomed him “Both my Grandma and Grandpa would be disgusted by their parish That is what it means to be Catholic.” Lengerich’s offer of dialogue and conversation falls flat when framed wihin the context of the priest denying Hakes the opportunity to honor his deceased loved one Why didn’t he enter into dialogue and conversation before making a decision It  is particularly disturbing that Lengerich somehow dug up a years-old photo of Hakes and then seems to have inferred from it that Hakes was in a same-gender relationship there are more productive uses for Lengerich’s time and energy as a priest Once again, a priest who should be a source of consolation and unity has added to a grieving family’s pain and divided a parish community. Denying LGBT people the ability to participate in mourning rituals or denying them Communion at a funeral Mass are not infrequent events sadly If church ministers cannot even be merciful and welcoming in these most painful moments how can the church expect LGBT people and their families to show up at any other moment I look at the bishops who have put that relatively young priest in the no-win situation he is in He should be strong enough to stand up for his parishioners but the universe that has formed him and that he views the world from haven’t empowered him to do it Lengerich could be struggling with his own sexual attraction It really is shameful that rigid rule followers play such a vicious role at times when mercy and respect for the dignity of the human being is called for This is precisely why the Church is watching the pews getting emptier by the year Lengerich ( not Father Lengerich ) is surprisingly young for such a negative tone are missing the big picture and narrowing their focus to Taliban like purity I hope Lengerich has an epiphany of some sort or maybe decides politics is his real calling My intuition impishly suggests to me that the Very Reverend Fr Bob Lengerich has some deep-seated internalized homophobia that he has yet to acknowledge This can be the only rational explanation for his violent behaviour towards Connor Hakes For a self-professed Christian to maltreat another human being when they are mourning the passing of a loved one beggars belief I wonder how he interprets Jesus’ Sermon on the Mount or Jesus’ parables (such as the good Samaritan) let alone the idea that what you did to the least of these you did to Jesus himself I pray that Fr Bob will learn to love himself and allow God to work some Divine Therapy on his heart of stone Ezekiel comes to mind … “I will give you a new heart and put a new spirit in you; I will remove from you your heart of stone and give you a heart of flesh.” Has his bishop responded publicly to the complaints “I have no desire to make windows into men’s souls.” but apparently some priests do Don’t ask the question if you don’t want to know the answer because once you start looking for sins you will find them for all I am not meaning to imply he is a sinner for his orientation or in my mind any actions related to it but that there are plenty of sins as some are defined to be found Another family of long Catholic tradition will probably now be looking for another church Does anyone wonder why the exit from this denomination is in full rush mode Should have read “loves so tenderly.” Please have this vicious prelate explain how someone singing at his grandmother’s funeral causes a scandal to the church What he is really saying is that gay folks must be so quiet about their sexuality and their life affirming relationships that they disappear into the back pew if they want to participate in Christ’s church Longerich regarding his insulting letter he sent to Mr The Catholic church has driven thousands of gay people away from the church because of their unbending willingness to accept us The church must face reality and make changes where all people are accepted with love and respect Just when I think our Church may be making baby steps toward becoming a more open and hospitable place there is yet another example of hateful (not too strong a word) treatment of a grieving young man paying respects to his beloved Grandmother The priest is in need of counseling (and perhaps a few more members of the Church are probably DONE in putting up with a distinctly non-Christian pastoral presence My condolences to the young man in this story he knew he had the unconditional love of his grandparents This priest is typical of the way the mean-spirited Ft I am from Indiana and went to the University of Notre Dame which is within the diocese the local bishops have tried to make the university a conservative enclave we are seeing the rise of a Catholic version of the Taliban The hierarchy doesn’t understand the love of the Gospels only the all-important rules to protect them from scandal I am bemused by the appeal to avoid scandal which they have made so evident I suspect everyone knew the grandson was gay and would not have objected to his singing for his grandmother’s funeral but now the ugliness of the priest’s stance reveals the real scandal Lengerich (who notably follows only right-wing politicians on twitter) was apparently preaching from the pulpit that it was a sin to vote for Hillary Clinton because of her abortion stance and apparently Lengerich believes that revenge is a dish best served cold Lengerich’s Facebook page sets most people’s gaydar pinging especially his photo with his “cousin” the boxer I think it’s clear there are some deep seated issues of self-hatred being played out here whether clergy or laity were allowed to celebrated and receive the sacraments This is indeed so sad and it is clear test this priest needs help Tragic though that there are so many like him It is also more than clear that Francis’ pastoral message and style are not being accepted Two things struck me about this article: 1) Father Lengerich “concluded the letter saying the parish did want Hakes present and did ‘want to enter into a real dialogue and conversation.'” And 2) “Hakes claimed that Fr Lengerich based his claims about the gay man’s sexual life on a picture posted to Facebook several years ago of Hakes celebrating Pride.” Assuming this report is accurate Why didn’t the pastor talk with the grandson personally And what was he doing checking the grandson’s Facebook page I really don’t understand why one’s sexual orientation or sexual behavior (whether real or perceived) has become such a central tenet (not “tenant,” as the pastor wrote) of the Catholic religion Funerals are extraordinarily sensitive times for those closed to the deceased I would not be surprised if this man never sets foot in this church again And the words in the letter: “You are loved by your Heavenly Father and by your Church Community We want you here…” ring quite hollow and discordant I sent him a pretty terse email about his pathetic decision This should go all the way TO POPE FRANCIS!!!! This entire narrative report (and our readers’ responses) need to be emailed directly to his bishop — for whatever good that may accomplish If the diocesan bishop himself is complicit in this priest’s attitude of “jihad” then I’m afraid the only remaining recourse would be to try to get through to Pope Francis himself through the Vatican’s media channels There’s no way I can see Pope Francis himself supporting this sort of counter-pastoral behavior by a Catholic priest This young priest is probably very well-meaning but hopefully he will get better guidance from his bishop about the welcoming attitude of Jesus than he has obviously had till now No wonder it is getting to be such a challenge for many of us who love our Catholic Faith to stay in an institution which tolerates such behavior toward a member of the Mystical Body and so grateful for what occurred at my sister in law’s funeral last summer but the funeral Mass was in a New England parish So many non-Catholics and others commented on the richness of the Mass and the pastor’s presence This is what “evangelizing” can be The Pharisees are alive and well I see… I am a young adult experiencing Same Sex Attractions and to read articles like this really I am glad that we are talking about ‘homosexual people’ because before all else comes the individual person And people should not be defined only by their sexual tendencies: let us not forget that God loves all his creatures and we are destined to receive his infinite love.” I also think the Church needed a pontiff like Francis I have friendships in Europe who have came back to the Chruch since Francis has been pontiff a State that I remember with rather less than affection over the Sylvia Likens affair A girl who was tortured for over a year by a self rightious woman and her family .And it seems there are other people here thaht enjoy the noriety of persecution That anyone should be so uneducated as to humiliate a homosexual man in this day and age is unbelievable […] Source: https://newwaysministryblog.wordpress.com/2016/11/30/priest-bans-gay-man-from-singing-at-grandmother… […] and website in this browser for the next time I comment Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply NEW RICHMOND – Judy Middeler’s first season coaching the swimmers at New Richmond High School came after Sparky Anderson won his last World Series title with the Cincinnati Reds Middeler is back to guide the team in and away from the pool Her red school shirt reads “Coach Judy,” but there’s no need to identify who is in charge Middeler’s voice resonates above all others in the natatorium and the swimmers seek her out before and after each race for inspiration she leads the Lady Lions who finished last season at 7-2 and in fourth place in the Southern Ohio Swim League Girls to watch this season are Claire Burns “I like how hard the swimmers are working,” Middeler said Taking over New Richmond’s boys for the late Joe Middeler is Rick Mahan after seven years as the middle school swim coach Mahan inherits a group of Lions that won nine times and finished second in the SOSL Several of the boys come from the Lions’ football team where Mahan also assists “It’s a great way to stay in shape,” he said They return home after the first of the year Jan The Glen Este boys swim team is led by junior John Martino who made Eastern Cincinnati Conference honorable mention a year ago in the 200 freestyle The girls squad was led by Olivia Killebrew who also made honorable mention in the 200 freestyle as a sophomore Taylor Cecil and Breanna Ruschman are Glen Este seniors Among the meets ahead for Trojans is the annual Milford Invitational Jan Lisa Werwinski is the new Glen Este dive coach and will focus on the flips of Bethany Berger “This is the first time Glen Este had had divers in a number of years,” Werwinski said “I’m very excited to work with these three girls to develop a program!” Amelia High School has reintroduced swimming after a four-year absence No other information was available at presstime McNicholas will be one of the smaller teams in the city with 10 total swimmers head coach Tessa Lengerich said it’s about quality instead of quantity who got into organized swimming as a freshman he started swimming with the Mercy Healthplex club team “He’s never had a bad day in the pool,” Lengerich said “I expect him to keep dropping his times.” She is also looking for big things from freshman Nick Rosenbaum He is a versatile swimmer who can swim the IM or middle distances a two-year state qualifier in the 500 free and a 2014 qualifier in the 200 free will handle most of the leadership duties for the Rockets’ girls’ team which doesn’t have a senior The Rockets will also get contributions from sophomore Skye Lewis and freshman Natalie Martinez Lengerich said Lewis will swim the breaststroke and should challenge the school record in the 100 the coach said she should be a strong asset in the team’s relay.