University of Oxford Momentum Award – Dementias
Lead Research Organisation:
University of Oxford
Department Name: UNLISTED
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
Technical Summary
Research and drug discovery for Alzheimer’s disease (AD) is undergoing a minor crisis with a
paucity of novel therapeutic approaches that is human evidence-based. Whilst, human genetic
studies have identified AD associated variants important for CNS glial function, there is
uncertainty over whether inflammation plays a beneficial or deleterious role. As a result, many
pharma companies are hesitant before committing significant resources to this novel therapeutic
opportunity. Our proposal is therefore focused on the scientific theme “Microglial interactions in
Alzheimer’s Disease”. The indicative projects will leverage four key capabilities within the
University of Oxford that represent untapped potential and are ripe for development for dementia
research, (1) access to human primary CNS cell types from human tissue coupled with single
cell analysis, (2) Human iPSC derived microglia, (3) High order imaging analysis of cellular
phenotypes driven by machine learning, (4) Protein-protein interaction disruption to probe
signalling pathways. The proposed projects approaches neuro-inflammation in AD through these
technical capabilities and are supported by world-leading bioinformatics and unrivalled structural
genomics consortium (SGC) pharmacological tools. These tools enable pharmacological pilot
studies within the projects, which if successful, will more rapidly lead to drug discovery projects
because of the enabling structural knowledge and chemical starting points held within the SGC.
Momentum funds will support the recruitment of a new research leader from outside the UK, as
well as supporting early career high flyers within Oxford. Our pharma partners and institution
leverages the MRC funds with significant institutional funds, platforms, space and expertise
totalling ~£2million (~£850K from the institution and $1.5million industry in-kind). We have
developed strong partnerships with Oxford University Hospital NHS Trust and industry including
world-leaders in single cell biology and dementia drug discovery. The Award will support the
diversification of target identification and develop human in vitro models that are more likely to
be predictive of clinical outcomes compared to current dominant murine models. Our approach
will facilitate synergy between disciplines and maximise the contribution of the grant towards the
acceleration of new dementia research in Oxford.
paucity of novel therapeutic approaches that is human evidence-based. Whilst, human genetic
studies have identified AD associated variants important for CNS glial function, there is
uncertainty over whether inflammation plays a beneficial or deleterious role. As a result, many
pharma companies are hesitant before committing significant resources to this novel therapeutic
opportunity. Our proposal is therefore focused on the scientific theme “Microglial interactions in
Alzheimer’s Disease”. The indicative projects will leverage four key capabilities within the
University of Oxford that represent untapped potential and are ripe for development for dementia
research, (1) access to human primary CNS cell types from human tissue coupled with single
cell analysis, (2) Human iPSC derived microglia, (3) High order imaging analysis of cellular
phenotypes driven by machine learning, (4) Protein-protein interaction disruption to probe
signalling pathways. The proposed projects approaches neuro-inflammation in AD through these
technical capabilities and are supported by world-leading bioinformatics and unrivalled structural
genomics consortium (SGC) pharmacological tools. These tools enable pharmacological pilot
studies within the projects, which if successful, will more rapidly lead to drug discovery projects
because of the enabling structural knowledge and chemical starting points held within the SGC.
Momentum funds will support the recruitment of a new research leader from outside the UK, as
well as supporting early career high flyers within Oxford. Our pharma partners and institution
leverages the MRC funds with significant institutional funds, platforms, space and expertise
totalling ~£2million (~£850K from the institution and $1.5million industry in-kind). We have
developed strong partnerships with Oxford University Hospital NHS Trust and industry including
world-leaders in single cell biology and dementia drug discovery. The Award will support the
diversification of target identification and develop human in vitro models that are more likely to
be predictive of clinical outcomes compared to current dominant murine models. Our approach
will facilitate synergy between disciplines and maximise the contribution of the grant towards the
acceleration of new dementia research in Oxford.
Publications
Zambon F
(2019)
Cellular a-synuclein pathology is associated with bioenergetic dysfunction in Parkinson's iPSC-derived dopamine neurons.
in Human molecular genetics
Xie C
(2019)
Culprit or Bystander: Defective Mitophagy in Alzheimer's Disease.
in Frontiers in cell and developmental biology
Watson L
(2018)
A Simplified Method for Generating Purkinje Cells from Human-Induced Pluripotent Stem Cells
in The Cerebellum
Volpato V
(2018)
Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.
in Stem cell reports
Sandor C
(2017)
Whole-exome sequencing of 228 patients with sporadic Parkinson's disease.
in Scientific reports
Sandor C
(2017)
Transcriptomic profiling of purified patient-derived dopamine neurons identifies convergent perturbations and therapeutics for Parkinson's disease.
in Human molecular genetics
Pokhilko A
(2021)
Global proteomic analysis of extracellular matrix in mouse and human brain highlights relevance to cerebrovascular disease.
in Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
Parkes I
(2018)
Neurovascular dysfunction in dementia - human cellular models and molecular mechanisms.
in Clinical science (London, England : 1979)
Nizami S
(2019)
Microglial inflammation and phagocytosis in Alzheimer's disease: Potential therapeutic targets.
in British journal of pharmacology
| Title | Additional file 2 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 2. : Video 1. Time-lapse video of dead SH-SY5Y phagocytosis assay. WT pMac phagocytosing pHrodo-labelled fixed SH-SY5Ys, displaying an increase in red fluorescence of the SH-SY5Ys after engulfment. In 96wp, 2 x 104 pMac were given 2 x 104 SH-SY5Ys, and images taken every 10 minutes at 20x for 3 hours, using an EVOS FL Auto automated microscope. Video is 2 frames per second. |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2020 |
| URL | https://springernature.figshare.com/articles/media/Additional_file_2_of_TREM2_Alzheimer_s_variant_R4... |
| Title | Additional file 2 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 2. : Video 1. Time-lapse video of dead SH-SY5Y phagocytosis assay. WT pMac phagocytosing pHrodo-labelled fixed SH-SY5Ys, displaying an increase in red fluorescence of the SH-SY5Ys after engulfment. In 96wp, 2 x 104 pMac were given 2 x 104 SH-SY5Ys, and images taken every 10 minutes at 20x for 3 hours, using an EVOS FL Auto automated microscope. Video is 2 frames per second. |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2020 |
| URL | https://springernature.figshare.com/articles/media/Additional_file_2_of_TREM2_Alzheimer_s_variant_R4... |
| Title | Additional file 3 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 3. : Video 2. Time-lapse video of synaptosome phagocytosis assay. WT pMac phagocytosing pHrodo-labelled synaptosomes, displaying an increase in red fluorescence of the synaptosomes after engulfment. In 96wp, 2 x 104 pMac were given 1 µg of synaptosomes, and images taken every 10 minutes at 20x for 3 hours, using an EVOS FL Auto automated microscope. Video is 2 frames per second. |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2020 |
| URL | https://springernature.figshare.com/articles/media/Additional_file_3_of_TREM2_Alzheimer_s_variant_R4... |
| Title | Additional file 3 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 3. : Video 2. Time-lapse video of synaptosome phagocytosis assay. WT pMac phagocytosing pHrodo-labelled synaptosomes, displaying an increase in red fluorescence of the synaptosomes after engulfment. In 96wp, 2 x 104 pMac were given 1 µg of synaptosomes, and images taken every 10 minutes at 20x for 3 hours, using an EVOS FL Auto automated microscope. Video is 2 frames per second. |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2020 |
| URL | https://springernature.figshare.com/articles/media/Additional_file_3_of_TREM2_Alzheimer_s_variant_R4... |
| Description | EU H2020 |
| Amount | € 18,000,000 (EUR) |
| Organisation | European Commission |
| Department | Innovative Medicines Initiative (IMI) |
| Sector | Public |
| Country | Belgium |
| Start | 09/2018 |
| End | 09/2023 |
| Description | IM2PACT |
| Amount | € 9,000,000 (EUR) |
| Funding ID | 807015 |
| Organisation | European Union |
| Sector | Public |
| Country | European Union (EU) |
| Start | 11/2018 |
| End | 10/2023 |
| Description | Targeting circadian mechanism for novel chronic pain treatments |
| Amount | £672,294 (GBP) |
| Funding ID | MR/X010864/1 |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2023 |
| End | 01/2026 |
| Title | IPSC microglia toolkit |
| Description | A NF kappa beta reporter human iPSC line that can be differentiated into various cells to report on this gene activation eg. in response to TNF alpha |
| Type Of Material | Cell line |
| Year Produced | 2018 |
| Provided To Others? | Yes |
| Impact | No impact yet |
| Title | Additional file 1 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 1 : Figure S1. Validation of R47H genotype. (A) CRISPR single guide RNA used for insertion of R47H mutation by Bioneer. (B) Chromatograms from sequencing of WT line BIONi010-C and R47H TREM2 line BIONi010-C-7. Red asterisk indicates the R47H mutation, black asterisks are silent mutations added by Bioneer to prevent re-cutting. Figure S2. SNP microarray of iPSCs. Chromosome karyograms from Illumina microarray SNP analysis, showing (A) BIONi010-C line, (B) BIONi010-C-7 R47H TREM2 line, (C) BIONi010-C-17 TREM2 KO line. Figure S3. Validation of R47H TREM2 and TREM2 KO pMac. (A) Macrophage surface markers CD11b, CD14, and CD45 measured by flow cytometry. Median fluorescence intensity (MFI) for each sample was normalized to the relevant isotype IgG, and then to the average for the three genotypes. Histogram shows means ± SEM, for n=3-4 harvests. 1-way ANOVA with Dunnett's post-hoc test, comparisons to WT line. ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant. (B) Total levels of TREM2 protein shown in a representative western blot (WB). (C-D) Surface TREM2 measured by immunofluorescence staining (IF): live pMac were stained with TREM2 antibody, followed by fluorescent secondary antibody, and subsequently fixed. Images are maximum projections from a z-stack of 5 slices, 1-5 µm, taken on an Opera Phenix microscope (Perkin Elmer). Quantified mean fluorescence (per µm2), for triplicate wells, was normalised to the average for the three genotypes, and then expressed as a ratio of whole-cell TREM2 staining from separate permeabilised wells on the same plate (D). Means ± SEM, for N=3 harvests, p = 0.047 in one-tailed paired t-test. (E-F) Kinetics of pMac calcium responses to 0.5 mM ATP (E), and 10 µg/mL TREM2 antibody (F). Means ± SEM, for N=3-5 harvests. Figure S4. Validation of antibodies for TREM2 immunocytochemistry. Fixed and permeabilized WT, R47H, and TREM2 KO pMac were stained for 1 hour at RT with three different TREM2 antibodies at the concentrations indicated, followed by staining with Alexa Fluor 488-conjugated secondary antibody (1:1000, Invitrogen). Cells were counterstained with DAPI nuclear dye and imaged on an EVOS FL Auto automated microscope (Thermo Fisher). Ab209814 showed cytoplasmic staining in all three genotypes, 13,483-1-AP showed nuclear staining in all three genotypes, whereas AF1828 stained cytoplasm and plasma membrane in WT and R47H TREM2 pMac but not TREM2 KO pMac. Scale bar is 100 µm. Figure S5. Validation of dead SH-SY5Y phagocytosis assay. (A) Freshly-fixed SH-SY5Ys stain uniformly for phosphatidylserine exposure (annexin V-FITC), but have limited cell permeability (propidium iodide). Live SH-SY5Ys do not stain for annexin V-FITC or propidium iodide, except for focal staining present on the few dead cells in culture. (B) No TREM2 expression in an SH-SY5Y not undergoing phagocytosis, marked with a white arrow. (C) No RAB9 expression in non-engulfed SH-SY5Ys, marked with a white arrow. (D) Dose-dependent uptake of dead SH-SY5Ys after 5 hours of phagocytosis with WT line BIONi010-C, means quantified from three independent experiments for % of spot positive (phagocytic) cells per well. Means ± SEM, for N=3 harvests. (E) Phagocytosis of 3 hours is inhibited with 10 µM cytochalasin D, 1 µM bafilomycin A1, 1 µM jasplakinolide, all with 1 hour pre-treatment, and 13 µg/mL recombinant annexin V added simultaneously to the dead SH-SY5Ys. Data was normalized to mean for each genotype per experiment. Means ± SEM, for N=3-6 harvests and with two WT cell lines (SFC840-03-03, the characterisation of this line is described in Fernandes et al [32], and BIONi010-C). 1-way ANOVA with Dunnett's post-hoc test, comparisons to untreated cells. * p < 0.05, *** p < 0.001. Figure S6. Validation of synaptosome phagocytosis assay. (A) Two whole synaptosomes surrounded by cell debris in the cryopreserved prep, visualised by negative staining electron microscopy. White asterisks label the pre-synaptic termini, with many pre-synaptic vesicles, whereas purple asterisks label the post-synaptic termini. A dark post-synaptic density can be seen between connected pre- and post-synaptic termini. (B) Synaptosomes stain uniformly for phosphatidylserine exposure (annexin V-FITC), comparison is with unstained synaptosomes. An area magnified by 5X is shown inset. (C) Dose-dependent uptake of dead SH-SY5Ys after 3 hours of phagocytosis with WT line BIONi010-C, reaching saturation above 30 µg. (D) Phagocytosis in BIONi010-C pMac is inhibited by 10 µM cytochalasin D and 1 µM bafilomycin A1, and increased by prior opsonisation of synaptosomes for 30 minutes with 20% human serum. Data was normalized to mean for each genotype per experiment, and is represented as sum of spot areas (µm2) per cell. Means ± SEM, for N=3-4 harvests. 1-way ANOVA with Dunnett's post-hoc test, comparisons to untreated cells. * p < 0.05, ** p < 0.01. Figure S7. Validation for cytokine ELISAs and transwell chemotaxis assay. Cytokine ELISAs: (A) Secretion of TNF in response to 4 hours of 0.1-1 µg/mL LPS. (B) Secretion of IL-6 in the same supernatants as (A). Means ± SEM, for N=3 harvests. 2-way ANOVA with Dunnett's post-hoc test. Comparisons with the coloured annotations are stimulations versus untreated cells (None) for each genotype. Comparisons with the black annotations are R47H or KO versus the WT line for each stimulation, all unannotated comparisons are not significant. Transwell chemotaxis assay: (C) Migration of WT pMac in transwell chemotaxis assay in the presence of four concentrations of ADP or C5a, for 6 hours. (D) Migration of WT pMac for 6 hours in the presence of 30 µM ADP is attenuated by 30 minutes pre-treatment with a P2RY12-selective inhibitor (PSB0739), but not a P2RY1 (MRS2179) or P2RY13 (MRS2211) inhibitor. (E) Migration of WT pMac for 6 hours in the presence of 3 nM C5a is attenuated by 30 minutes pre-treatment with a C5aR inhibitor (PMX-53), or a Syk inhibitor (OXSI-2, 3 µM). Means ± SEM, for N=3-4 harvests. 1-way ANOVA with Dunnett's post-hoc test, pairwise comparisons to control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure S8. RNA-seq differentially-expressed genes (DEGs). Volcano plots shown for DEGs relative to WT: (A) TREM2 KO and (B) R47H TREM2 pMac. Dashed lines show cut-offs at log2-fold-change=2 and p=0.001. Enrichment of Gene Ontology (GO) terms in significant (adjusted p value <0.05) DEGs relative to WT: (C) TREM2 KO, and (D) R47H TREM2 pMac. Top 30 terms shown in order of the adjusted p value, the relative R-score represents the average direction of change. Circle size corresponds with number of DEGs. Figure S9. Flow cytometry of integrins. Representative flow cytometry histograms for Figure 6e. Figure S10. Proportions of TREM2 transcript splice variants. TREM2 differential transcript usage analysis was performed on the RNA-seq data with the DRIMSeq bioconductor package, using Salmon-quantified transcript counts. DRIMSeq assumes an Dirichlet Multinomial model (DM) for each gene, where the total count for the gene is considered fixed, and the quantity of interest is the proportion for the transcript within a gene for each sample. A likelihood ratio test was used to test for gene and transcript level DTU between R47H vs WT, and no significant difference in transcript usage was found. Transcripts are described in Del-Aguila et al [62]: ENST00000373113 is the canonical TREM2 transcript and the longest, with five exons; ENST00000373122 is the second longest and lacks the 5' exon but includes the transmembrane domain; ENST00000338469 is the shortest and excludes the transmembrane domain. The bars represent three sequential ages of iPSC-macrophages. Figure S11. Correlation of TREM2 KO DEGs with Claes et al (2019). Log-2-fold change values for the significant DEGs (adjusted p value <0.05) of the current study (purple) plotted against significant DEGs from Claes et al (orange). Overlapping significant DEGs shown in green. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_1_of_TREM2_Alzheimer_s_variant_... |
| Title | Additional file 1 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 1 : Figure S1. Validation of R47H genotype. (A) CRISPR single guide RNA used for insertion of R47H mutation by Bioneer. (B) Chromatograms from sequencing of WT line BIONi010-C and R47H TREM2 line BIONi010-C-7. Red asterisk indicates the R47H mutation, black asterisks are silent mutations added by Bioneer to prevent re-cutting. Figure S2. SNP microarray of iPSCs. Chromosome karyograms from Illumina microarray SNP analysis, showing (A) BIONi010-C line, (B) BIONi010-C-7 R47H TREM2 line, (C) BIONi010-C-17 TREM2 KO line. Figure S3. Validation of R47H TREM2 and TREM2 KO pMac. (A) Macrophage surface markers CD11b, CD14, and CD45 measured by flow cytometry. Median fluorescence intensity (MFI) for each sample was normalized to the relevant isotype IgG, and then to the average for the three genotypes. Histogram shows means ± SEM, for n=3-4 harvests. 1-way ANOVA with Dunnett's post-hoc test, comparisons to WT line. ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant. (B) Total levels of TREM2 protein shown in a representative western blot (WB). (C-D) Surface TREM2 measured by immunofluorescence staining (IF): live pMac were stained with TREM2 antibody, followed by fluorescent secondary antibody, and subsequently fixed. Images are maximum projections from a z-stack of 5 slices, 1-5 µm, taken on an Opera Phenix microscope (Perkin Elmer). Quantified mean fluorescence (per µm2), for triplicate wells, was normalised to the average for the three genotypes, and then expressed as a ratio of whole-cell TREM2 staining from separate permeabilised wells on the same plate (D). Means ± SEM, for N=3 harvests, p = 0.047 in one-tailed paired t-test. (E-F) Kinetics of pMac calcium responses to 0.5 mM ATP (E), and 10 µg/mL TREM2 antibody (F). Means ± SEM, for N=3-5 harvests. Figure S4. Validation of antibodies for TREM2 immunocytochemistry. Fixed and permeabilized WT, R47H, and TREM2 KO pMac were stained for 1 hour at RT with three different TREM2 antibodies at the concentrations indicated, followed by staining with Alexa Fluor 488-conjugated secondary antibody (1:1000, Invitrogen). Cells were counterstained with DAPI nuclear dye and imaged on an EVOS FL Auto automated microscope (Thermo Fisher). Ab209814 showed cytoplasmic staining in all three genotypes, 13,483-1-AP showed nuclear staining in all three genotypes, whereas AF1828 stained cytoplasm and plasma membrane in WT and R47H TREM2 pMac but not TREM2 KO pMac. Scale bar is 100 µm. Figure S5. Validation of dead SH-SY5Y phagocytosis assay. (A) Freshly-fixed SH-SY5Ys stain uniformly for phosphatidylserine exposure (annexin V-FITC), but have limited cell permeability (propidium iodide). Live SH-SY5Ys do not stain for annexin V-FITC or propidium iodide, except for focal staining present on the few dead cells in culture. (B) No TREM2 expression in an SH-SY5Y not undergoing phagocytosis, marked with a white arrow. (C) No RAB9 expression in non-engulfed SH-SY5Ys, marked with a white arrow. (D) Dose-dependent uptake of dead SH-SY5Ys after 5 hours of phagocytosis with WT line BIONi010-C, means quantified from three independent experiments for % of spot positive (phagocytic) cells per well. Means ± SEM, for N=3 harvests. (E) Phagocytosis of 3 hours is inhibited with 10 µM cytochalasin D, 1 µM bafilomycin A1, 1 µM jasplakinolide, all with 1 hour pre-treatment, and 13 µg/mL recombinant annexin V added simultaneously to the dead SH-SY5Ys. Data was normalized to mean for each genotype per experiment. Means ± SEM, for N=3-6 harvests and with two WT cell lines (SFC840-03-03, the characterisation of this line is described in Fernandes et al [32], and BIONi010-C). 1-way ANOVA with Dunnett's post-hoc test, comparisons to untreated cells. * p < 0.05, *** p < 0.001. Figure S6. Validation of synaptosome phagocytosis assay. (A) Two whole synaptosomes surrounded by cell debris in the cryopreserved prep, visualised by negative staining electron microscopy. White asterisks label the pre-synaptic termini, with many pre-synaptic vesicles, whereas purple asterisks label the post-synaptic termini. A dark post-synaptic density can be seen between connected pre- and post-synaptic termini. (B) Synaptosomes stain uniformly for phosphatidylserine exposure (annexin V-FITC), comparison is with unstained synaptosomes. An area magnified by 5X is shown inset. (C) Dose-dependent uptake of dead SH-SY5Ys after 3 hours of phagocytosis with WT line BIONi010-C, reaching saturation above 30 µg. (D) Phagocytosis in BIONi010-C pMac is inhibited by 10 µM cytochalasin D and 1 µM bafilomycin A1, and increased by prior opsonisation of synaptosomes for 30 minutes with 20% human serum. Data was normalized to mean for each genotype per experiment, and is represented as sum of spot areas (µm2) per cell. Means ± SEM, for N=3-4 harvests. 1-way ANOVA with Dunnett's post-hoc test, comparisons to untreated cells. * p < 0.05, ** p < 0.01. Figure S7. Validation for cytokine ELISAs and transwell chemotaxis assay. Cytokine ELISAs: (A) Secretion of TNF in response to 4 hours of 0.1-1 µg/mL LPS. (B) Secretion of IL-6 in the same supernatants as (A). Means ± SEM, for N=3 harvests. 2-way ANOVA with Dunnett's post-hoc test. Comparisons with the coloured annotations are stimulations versus untreated cells (None) for each genotype. Comparisons with the black annotations are R47H or KO versus the WT line for each stimulation, all unannotated comparisons are not significant. Transwell chemotaxis assay: (C) Migration of WT pMac in transwell chemotaxis assay in the presence of four concentrations of ADP or C5a, for 6 hours. (D) Migration of WT pMac for 6 hours in the presence of 30 µM ADP is attenuated by 30 minutes pre-treatment with a P2RY12-selective inhibitor (PSB0739), but not a P2RY1 (MRS2179) or P2RY13 (MRS2211) inhibitor. (E) Migration of WT pMac for 6 hours in the presence of 3 nM C5a is attenuated by 30 minutes pre-treatment with a C5aR inhibitor (PMX-53), or a Syk inhibitor (OXSI-2, 3 µM). Means ± SEM, for N=3-4 harvests. 1-way ANOVA with Dunnett's post-hoc test, pairwise comparisons to control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure S8. RNA-seq differentially-expressed genes (DEGs). Volcano plots shown for DEGs relative to WT: (A) TREM2 KO and (B) R47H TREM2 pMac. Dashed lines show cut-offs at log2-fold-change=2 and p=0.001. Enrichment of Gene Ontology (GO) terms in significant (adjusted p value <0.05) DEGs relative to WT: (C) TREM2 KO, and (D) R47H TREM2 pMac. Top 30 terms shown in order of the adjusted p value, the relative R-score represents the average direction of change. Circle size corresponds with number of DEGs. Figure S9. Flow cytometry of integrins. Representative flow cytometry histograms for Figure 6e. Figure S10. Proportions of TREM2 transcript splice variants. TREM2 differential transcript usage analysis was performed on the RNA-seq data with the DRIMSeq bioconductor package, using Salmon-quantified transcript counts. DRIMSeq assumes an Dirichlet Multinomial model (DM) for each gene, where the total count for the gene is considered fixed, and the quantity of interest is the proportion for the transcript within a gene for each sample. A likelihood ratio test was used to test for gene and transcript level DTU between R47H vs WT, and no significant difference in transcript usage was found. Transcripts are described in Del-Aguila et al [62]: ENST00000373113 is the canonical TREM2 transcript and the longest, with five exons; ENST00000373122 is the second longest and lacks the 5' exon but includes the transmembrane domain; ENST00000338469 is the shortest and excludes the transmembrane domain. The bars represent three sequential ages of iPSC-macrophages. Figure S11. Correlation of TREM2 KO DEGs with Claes et al (2019). Log-2-fold change values for the significant DEGs (adjusted p value <0.05) of the current study (purple) plotted against significant DEGs from Claes et al (orange). Overlapping significant DEGs shown in green. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_1_of_TREM2_Alzheimer_s_variant_... |
| Title | Additional file 4 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 4 Results of RNA-seq differential gene expression analysis with DESeq2. DESeq2 output for R47H TREM2 vs WT and TREM2 KO vs WT, with gene name, relative expression ("Base Mean"), log-2-fold-change from the WT, and Benjamini-Hochberg-adjusted p values indicated. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_4_of_TREM2_Alzheimer_s_variant_... |
| Title | Additional file 4 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 4 Results of RNA-seq differential gene expression analysis with DESeq2. DESeq2 output for R47H TREM2 vs WT and TREM2 KO vs WT, with gene name, relative expression ("Base Mean"), log-2-fold-change from the WT, and Benjamini-Hochberg-adjusted p values indicated. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_4_of_TREM2_Alzheimer_s_variant_... |
| Title | Additional file 5 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 5. Results of RNA-seq Ingenuity Pathways Analysis on TREM2 KO PPI groups. For each PPI group separate files give information about the genes assigned to that group (suffix "_Genes"), the predicted upstream regulators with Activation Z-score indicating whether the change mimics (+) or opposes (-) TREM2 KO transcriptional effects (suffix "_UpstreamAnalysis"), and the relationship of predicted upstream regulators to disease/functions (suffix "_RegulatorEffects"). |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_5_of_TREM2_Alzheimer_s_variant_... |
| Title | Additional file 5 of TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages |
| Description | Additional file 5. Results of RNA-seq Ingenuity Pathways Analysis on TREM2 KO PPI groups. For each PPI group separate files give information about the genes assigned to that group (suffix "_Genes"), the predicted upstream regulators with Activation Z-score indicating whether the change mimics (+) or opposes (-) TREM2 KO transcriptional effects (suffix "_UpstreamAnalysis"), and the relationship of predicted upstream regulators to disease/functions (suffix "_RegulatorEffects"). |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_5_of_TREM2_Alzheimer_s_variant_... |
| Title | Molecular signatures of microglial activation |
| Description | 1. IPSC Microglia co-cultures with neurons were exposed to LPS and the single cell gene expression response was assessed using the 10X genomics platform 2. iPSC microglia were exposed to LPS or PGE2 +/- ATP followed by single cell gene expression using the 10X genomics platform |
| Type Of Material | Database/Collection of data |
| Year Produced | 2019 |
| Provided To Others? | Yes |
| Impact | None yet |
| Description | A genetically validated cellular atlas for pain |
| Organisation | Cardiff University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Expertise and methods in chronic pain disorders for electronic record analysis Single cell biology |
| Collaborator Contribution | Expertise and methods in chronic pain disorders for electronic record analysis |
| Impact | Real-world data analysis of chronic pain outcomes Novel genome wide association studies Manuscript in preparation |
| Start Year | 2021 |
| Description | A genetically validated cellular atlas for pain |
| Organisation | GlaxoSmithKline (GSK) |
| Department | Neuroscience (GSK) |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Expertise and methods in chronic pain disorders for electronic record analysis Single cell biology |
| Collaborator Contribution | Expertise and methods in chronic pain disorders for electronic record analysis |
| Impact | Real-world data analysis of chronic pain outcomes Novel genome wide association studies Manuscript in preparation |
| Start Year | 2021 |
| Description | IMI IM2PACT |
| Organisation | University of Oxford |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This 18 million euro programme is a public-private partnership to develop predictive in vitro and in vivo models of the Blood-Brain-Barrier to improve therapeutic access to the central nervous system. Cader is the Consortium coordinator; Webber is a work-package leader |
| Collaborator Contribution | There are multiple academic institutions and SMEs across Europe involved in this consortium as well large Pharma. |
| Impact | Collaboration is multi-disciplinary. Bioinformatics, stem cell models; pharmacokinetics; tissue engineering; preclinical models; nanotechnology |
| Start Year | 2018 |
| Description | Purinergic signalling in microglia-neuron interactions |
| Organisation | Orion Corporation |
| Country | Finland |
| Sector | Private |
| PI Contribution | Development of iPSC-microglia models and response to ATP; calcium imaging and multi-electrode array analysis of microglia and neutrons |
| Collaborator Contribution | Funding and experimental design |
| Impact | Assay development |
| Start Year | 2018 |
| Company Name | Human Centric DD |
| Description | Human Centric DD develops drugs that target neurological diseases using human data analysis. |
| Year Established | 2020 |
| Impact | N/A |
| Website | https://www.humancentricdd.com/ |
| Description | ARUK Dementia Research day talk |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited talk about the Momentum project and microglia for other Dementia Researchers, organised by Alzheimer's Research UK Oxford branch |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.dpag.ox.ac.uk/research/wade-martins-group/oxford-aruk-network-centre/research-day-2015 |
| Description | ARUK Oxford Network Research Day |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | Presentation to researchers at the University of Oxford, as well as attendees from local industry, sparking discussion of the projects and plans for further interaction with new research groups. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.dpag.ox.ac.uk/research/wade-martins-group/oxford-aruk-network-centre/research-day-2015 |
| Description | Banbury Center (Cold Spring Harbor Labs) Meeting |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | An expert group meeting to discuss key issues in using stem cell for neuropharmacology and studies of neurodegenerative disorders |
| Year(s) Of Engagement Activity | 2017 |
| Description | DPUK Talk |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | A talk on developing stem cell models and experimental medicine approaches for vascular health in dementia |
| Year(s) Of Engagement Activity | 2017 |
| Description | Interview on BBC Radio Wales |
| Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Media (as a channel to the public) |
| Results and Impact | Interview discussing how scientists in Wales are at the centre of health research, this interview focused on work aimed at identifying whether people are at risk of developing dementia |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.bbc.co.uk/programmes/b0b9zrvv |
| Description | OPDC Participants Day |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Patients, carers and/or patient groups |
| Results and Impact | Oxford Parkinson's Disease Centre Talk to study participants, patients and carers about microglia |
| Year(s) Of Engagement Activity | 2017 |
| URL | http://opdc.medsci.ox.ac.uk/podcasts-2017 |
| Description | PD UK patient forum talk |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Patients, carers and/or patient groups |
| Results and Impact | I gave a PD UK patient forum talk at Waterlooville Community Centre, 10 Maurepas Way, Waterlooville, Hampshire, PO7 7AY. Wed 12/07/2017 "How can skin cells help us find therapeutics for Parkinson's?" |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.parkinsons.org.uk/ |
| Description | Parkinson's UK Research Roadshow Cardiff |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | Cardiff edition of Parkinson's UK Research Roadshow took place at Hadyn Ellis Building. It was a UK wide event, inviting patients and family members to hear about exciting research taking place at specific locations and providing information on how involvement in research could help move towards better treatments and ultimately a cure. Interactive discussions with patients, carers and charity workers about 'Can stem cells find new treatments for Parkinson's' |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.healthandcareresearch.gov.wales/noticeboard/parkinsons-uk-research-roadshow-cardiff/?for... |
| Description | Pharma collaboration meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Around 15 delegates from J&J attended a meeting at the University of Oxford with research teams engaged in drug discovery for mental health. This was one of a series of meetings that led to the company being part of the MRC Oxford Mental Health Data Pathfinder project. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Presentation at opening of new Institute |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Supporters |
| Results and Impact | Around 250 people attended the opening of the Li Ka Shing Centre for Health Information and Discovery. 7 scientists showcased big data projects, sparking questions and discussion. |
| Year(s) Of Engagement Activity | 2017 |
| URL | http://www.ox.ac.uk/news/2017-05-16-world%E2%80%99s-largest-health-big-data-institute-opens-oxford |
| Description | Scientific Talk at King's College London |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | Academic research talk at external institution |
| Year(s) Of Engagement Activity | 2018 |
| Description | Stem Cell Outreach Workshop |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Hosted an activity in a workshop for senior school pupils to learn about human stem cells |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.path.ox.ac.uk/news/secondary-school-students-peek-real-life-world-stem-cell-research-dun... |
| Description | Talk at Basel Life Conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Invited talk about iPS-derived microglia at Basel Life Conference session: Pluripotent stem cells in disease modeling and drug discovery |
| Year(s) Of Engagement Activity | 2017 |
| Description | Talk at Cardiff Dementia Research Institute |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | Scientific talk to the Dementia Research Institute, Cardiff University |
| Year(s) Of Engagement Activity | 2018 |
| Description | Talk at Oxford Parkinson's Disease Centre Research Day |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | Talk at Oxford Parkinson's Disease Centre Research Day, which attracts delegates from across the region, including both resaerchers, clinicians and patients/carers |
| Year(s) Of Engagement Activity | 2018 |
| Description | Talk at industry meeting in Belgium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | A group from Oxford travelled to meet with Pharma in Belgium to discuss potential opportunities for collaboration, to be followed up. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Talk to Oxford Immunology Group |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited talk about microglia to the Oxford Immunology Group Neuroinflammation meeting |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://oig.kennedy.ox.ac.uk/ |
| Description | UK Dementia Research Seminar (Tokyo) |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | A foreign commonwealth office sponsored visit to Tokyo for 2 days of engagement with Japanese dementia researchers to discuss shared interests and possible collaborations |
| Year(s) Of Engagement Activity | 2017 |