Impact of iPSC-derived microglial exosomes on neurons: Role of TREM2 and implication in Alzheimer's Disease

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Salih, John Hardy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7487794/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microglial exosomes are key secretome components that modulate cell-to-cell communication mediating protective or detrimental effects depending on the environmental context upon release. The R47H variant in the microglial triggering receptor expressed on myeloid cells-2 (TREM2) increases the risk for late-onset Alzheimer’s disease (AD) and influences microglia function, contributing to neurodegeneration. Our group has previously shown that the proteome content of exosomes released by microglia harboring the TREM2 R47H mutation differs from that of TREM2 common variant microglial exosomes, suggesting an altered microglia-neuron interaction and neuronal function upon their secretion. To further investigate how R47H variants or TREM2 loss modifies exosome effects on neurons, we assessed further their effects on human iPSC-neurons. We assessed transcriptome and proteome changes in neurons using RNA-seq and proteomic analyses. Our findings reveal that exosomes secreted by R47H variant iPSC-microglia differentially regulate the neuronal transcriptome associated with metabolic pathways and synaptic function and the observed changes in the cell stress-related proteome of neurons further supports this. Additionally, we provide evidence regarding the effects of these exosomes on pre-synaptic and post-synaptic and apoptotic markers expressed by our neuronal model and the influence of TREM2 status on synaptic functioning. Collectively, our data contribute to the characterization of human microglial exosome functions and provide novel insights into how this emerging communication pathway is affected by TREM2 late-onset AD risk variants. exosomes microglia neurons TREM2 R47H AD risk variant neurodegeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main points Exosomes secreted by the R47H variant or TREM2 -/- iPSC-microglia differentially regulate the neuronal transcriptome. TREM2 play a role in modulating neuronal metabolism, neurite outgrowth and synaptic function via exosomal pathways. R47H and TREM2 -/- microglia-derived exosomes fail to protect neurons from synaptic dysfunction upon Aβ 1-42 challenge 1. Introduction Microglia, the immune cells of the brain, interact with nearly all brain cell types to support developmental processes, maintain homeostasis, contribute to tissue repair, and play a role in the pathogenesis of diseases (Gao et al., 2023). Microglial triggering receptor expressed on myeloid cells 2 (TREM2) regulates microglial functions including phagocytosis, metabolism, immune activation and recent evidence suggests that TREM2 plays a crucial role in microglia-neuron cross talk (reviewed in (Pocock et al., 2024). Rare TREM2 variants, including the loss-of-function (LoF) R47H variant, have been associated with an increased risk of late-onset Alzheimer’s Disease (AD), the most prevalent form of AD. Microglia expressing LoF TREM2 variants or those completely lacking TREM2 exhibit phagocytic, metabolic, immune activation deficits and affect neuronal health (Garcia-Reitboeck et al., 2018; Piers et al., 2020; Cosker et al., 2021; Popescu et al., 2023; Tagliatti et al., 2024). However, the pathways whereby TREM2-dependent microglia shape neuronal function, and how AD risk variants impact upon these pathways remain largely underexplored. Extracellular vesicles (EVs) are shed from cells and are nano-sized particles which can be classified broadly into exosomes (40-150 nm), microvesicles (50-2000 nm), or apoptotic bodies (50-5000 nm). Exosomes are small EVs, ranging from 40-200 nm in size originating from the endocytic pathway and carrying a cargo of nucleic acids, proteins, and lipids that vary, reflecting their cellular origin and state (Antonucci et al., 2012; Gupta and Pulliam, 2014; Yang et al., 2018; Mathieu et al., 2019). Exosomes are key microglia secretome components and once released are taken into recipient cells, where they facilitate intracellular communication and crosstalk with other brain cells, or even propagate disease states (Guo et al., 2021; Vasilopoulou et al., 2024b). We have previously demonstrated that the TREM2 LoF R47H variant results in an altered content of the exosomes released by human induced pluripotent stem cell (iPSC) - microglia, particularly with regard to metabolic and transcription pathway changes (Mallach et al., 2021a; b). At the same time, TREM2 has been shown to affect exosomal-mediated spreading and the seeding of pathological AD associated proteins (Leyns et al., 2019; Zhu et al., 2022). This evidence suggests that TREM2, which can be found in the exosomal content and membrane of the exosomes (Mallach et al., 2021a; b) can influence microglial intercellular communication pathways, affecting various pathological events and thereby contributing to disease progression. In this study we aimed to go deeper into the TREM2-dependent beneficial, or detrimental effects delivered by exosomes secreted by human iPSC-microglia harbouring the LoF R47H variant or lacking TREM2, through transcriptomic, proteomic and functional assessment of neurons. We hypothesise that TREM2 variant microglial exosomes will lead to changes to neuronal transcriptome translated into functional changes that may promote beneficial or detrimental neuronal processes in a TREM2-dependent manner. Our approach provides insights into the disease mechanisms underlying microglial-driven neurodegenerative processes in AD and other diseases characterized by neuronal dysfunction. 2. Materials and Methods 2.1 iPSC-neuronal culture The control iPSC line BIONi010-C (EBiSC) was used for the experiments. iPSCs were differentiated into cortical neurons as described in Shi et al., (2012 ) with slight modifications. Briefly, iPSC were grown to 100% confluence in E8 media, and neural induction started (Day 0) by switching media to neuronal induction media (NIM), which consisted of neuronal maintenance media (NMM) supplemented with 10μM SB-431542 (Sigma) and 200nM LDN-193189 (Sigma). NIM was replaced daily for 10-12 days until a homogeneous neuroepithelial sheet was formed. On day 12, neuroepithelial cells were passaged using dispase to laminin-coated plates (50μg/mL, Gibco), and the medium was switched to NMM (which consisted of 1:1 mixture of Dulbecco's modified eagle medium F12 (DMEM-F12) and Neurobasal supplemented with 0.5x B27, 0.5x N2, 1.7x GlutaMAX, 0.5x sodium pyruvate, 25μg/ml Pen/Strep, 50μM 2-mercaptoethanol, 0.5x nonessential amino acids, 2.5 μg/ml insulin. For the next 3 days, 20ng/ml bFGF was added to NMM to promote rosette expansion. On day 16, bFGF was withdrawn, and cells were maintained to NMM until day 25, changing the media every other day. After expansion, around days 23-25, neuronal progenitor cells (NPC) were passaged with Accutase (Sigma) to laminin-coated plates (Figure 1a). NMM was replaced every other day, and NPC were passaged every 2-4 days using Accutase until clear neuronal projections were formed. On day 33 after induction cells were dissociated with Accutase for the final time and plated in laminin (50μg/mL, Gibco) /poly-L-ornithine (PLO) (0.01% wt/vol, Sigma) coated plates, at a final density of 50,000 cells per cm 2 . When cells completed 95-100 days in culture, mature human iPSC-neurons were used for the experiments. To characterize cortical neuron cultures, iPSC-neurons were seeded and matured on 13 mm glass coverslips. On day 100, the cells were fixed with 4%PFA in Dulbecco's phosphate buffered saline (PBS) for 20 min at room temperature (RT) following permeabilisation with 0.1% Triton X. Unspecific binding sites were blocked with 5% normal goat serum for 1h at RT, and the cells were incubated with primary antibodies for staining of established neuronal markers, including Tuj1 (1:1000), NeuN (1:100), TBR1 (1:500) BRN2 (1:500), SATB2 (1:200), synaptophysin (1:500) at 4 o C overnight. The cells were then incubated with anti-mouse or anti-rabbit AlexaFluor 488 or 568 conjugated secondary antibodies (1:500) and nuclei were counterstained with DAPI. Different regions per coverslip were imaged on a Zeiss LSM710 confocal microscope (40X, 63X) using the LSM Pascal 5.0 software. Brightfield images were obtained at critical points of the neuronal differentiation using a Leica microscope (10X, 20X) ( Figure 1a) . All antibodies used are listed in supplementary table S1. 2.2 iPSC-derived microglia Two control iPSC lines were used: SFC840 (Stembancc), and BIONi010-C (EBiSC), to generate TREM2 common variant (Cv) microglia. R47H heterozygous fibroblasts were previously acquired with an MTA between University College London and University of California Irvine Alzheimer's Disease Research Center (UCI ADRC; Prof M Blurton-Jones) and fibroblast reprogramming and Karyotyping of a number of lines and clones described previously (Piers et al., 2020). Three clones from two ADRC R47H heterozygous (R47H het ) patient lines were used: ADRC8 (clones 3, 6, and 12), and ADRC26 (clones 3, 5, and 15). TREM2 R47H homozygous (R47H hom ; BIONi010-C7) and TREM2 knock-out (TREM2 -/- ; BIONi010-C17) were gene edited BioNi010-C, all purchased from EBiSC. All iPSC were maintained and routinely passaged in Essential 8 medium. iPSC-microglia were generated using our previously described protocol (Piers et al., 2020) which incorporates procedures from earlier protocols (Garcia-Reitboeck et al., 2018; Xiang et al., 2018). Briefly, embryoid bodies were generated from 70% confluent iPSC and maintained for 5 days in 100 ng/ml ROCK inhibitor, 50 ng/ml VEGF, 50 ng/ml BMP-4 and 20 ng/ml SCF. The embryoid bodies were then transferred into flasks where they were maintained in X-VIVO (Lonza) supplemented with 100 ng/ml MCSF and 25 ng/ml IL3 for further differentiation. After 4-5 weeks, progenitor cells were collected and plated in iPSC-microglia maintenance medium supplemented with 100 ng/ml IL-34, 25 ng/ml MCSF, and 5 ng/ml TGF-β. Medium was changed every week and 2 weeks after plating, 100 ng/ml CX3CL1 and 100 ng/ml CD200 were added to microglia medium (maturation medium) and subsequently to the microglia for 3 days to achieve final maturation ( Figure 1b). All growth factors were purchased from PeproTech (Thermo Fisher Scientific). 2.3 . Exosome collection and characterisation by Transmission Electron microscopy and Western Blot The iPSC-microglia were plated and matured on 6-well plates at a density of 50.000 cells/cm 2 . Medium was changed on the iPSC-microglia 48 h before the experiment. The supernatant was collected, centrifuged for 15 min at 300g (av) and subsequently used for exosome collection. Exosomes were extracted using an ExoQuick-TC kit (System Biosciences) by centrifugation of the supernatant for 15 min at 3000g (av) followed by ExoQuick solution addition overnight at 4 ο C. After two subsequent centrifugations at 1500g (av) for 30 min and 5 min, the pellets were collected and resuspended in PBS without Ca 2+/ Mg 2+ , pH 7.0–7.3 or RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Exosomal content was quantified by performing Micro BCA TM protein quantification (Invitrogen, Thermo Fisher Scientific) and used for neuronal treatments at a concentration of 1 μg/10,000 cells as described previously (Mallach et al., 2021a; b). Exosome samples for transmission electron microscopy (TEM) were dropped with a Pasteur pipette onto a carbon/formvar coated copper grid. After 15 seconds excess sample was blotted off with filter paper. Then a drop of 2% phosphotungstic acid stain was added and blotted after 15 seconds. The grid was placed into a specimen holder and inserted into a Phillips/FEI CM 120 BioTwin TEM for imaging at 80kV (Figure 1ci) . Exosomal marker expression was confirmed by western blot. Briefly, exosomal pellets or iPSC-microglia cells were lysed in RIPA buffer and centrifuged at 15,000g (av) for 15 min and the samples denatured and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and blocked with 5% milk in Tris buffered saline solution with 1% Tween -20 (TBS-T), and incubated with primary antibodies including ALIX (1:000), HSP70 (1:1000) and β-actin (1:5000) (Supplementary table S1) overnight at 4 o C followed by incubation with the appropriate HRP-conjugated secondary antibody at 1:5000 for 1h at RT (Figure 1cii). The membranes were washed 3x with TBS-T followed by a final TBS-T wash and visualized using the Odyssey detection system (LiCor). 2.4. RNA sequencing iPSC-neurons were plated at a density of 50,000 cells/cm 2 , and on day 100 the cells were incubated with iPSC-microglia exosomes for 48 h. After treatment, iPSC-neurons were lysed in RNA protection buffer (New England Biolabs) and used for RNA extraction following the manufacturer’s instructions (Monach Total RNA, New England Biolabs). RNA concentration and 260/230 260/280 ratios were measured by NanoDrop (DeNovix DS-11FX+spectophotometer). Quality of the total RNA was assessed using capillary electrophoresis.The RNA-seq library preparation and sequencing was performed by Novogene, UK. Eukaryotic RNA-seq libraries were prepared using the NEBNext® Ultra™ RNA Library preparation Kit, generating 250-300 bp cDNA inserts. Quantified libraries were pooled and sequenced by Illumina NovaSeq6000 generating 150-bp paired-end (PE150) reads, multiplex samples per lane - 9G data per sample. From FASTQ files, adaptors and low quality base pairs were removed. Transcripts were aligned with HISAT2 (Mortazavi et al., 2008), using gene annotation from human reference genome GRCh38/hg38. RNA-seq count data were normalized using the DESeqDataSetFromMatrix() function in DESeq2 (Love et al., 2014), with default parameters and a design formula ~ Batch + Genotype of origin of exosomes to model batch as a covariate during differential expression analysis. DESeq2 uses raw read counts, applies normalization, and estimates dispersion. The effect of genotype of the origin of exosomes was tested, and differentially expressed genes were stated as r value < 0.01 and fold-change ³ 0.5. For visualization using heatmaps, variance-stabilizing transformation was applied using vst(), and batch effects were further removed from the transformed data using limma::removeBatchEffect() with default settings. Biological annotations were identified against Gene Ontology, REACTOME (Fabregat et al., 2018) and KEGG databases using gProfileR2 (Kanehisa et al., 2016; Raudvere et al., 2019), using genes with r value < 0.01 and fold-change ³ 0.5, using all detected genes in our experiment as the background. 2.5. Real-time qPCR Quantitative qPCR experiments were performed to determine the effect of exosomes on iPSC-neurons and for RNA-seq validation. RNA was extracted as described in section 2.4 . cDNA was generated with a high-capacity cDNA reverse transcription kit (Applied Biosystems) and qPCR analysis was performed using Taqman Universal Master Mix (Life Technologies) using specific primers (Supplementary table 2) and the MxPro qPCR software (Agilent). Expression was normalized to GAPDH. 2.6. Seahorse Analysis For real-time analysis of oxygen consumption rates (OCR) we used the Seahorse XF Cell Mito Stress Test kit (Agilent Technologies) as previously described for our iPSC-microglia (Garcia-Reitboeck et al., 2018; Cosker et al., 2021; Vasilopoulou et al., 2024a). iPSC-neurons were plated at a density of 50.000 per cm 2 , on laminin/PLO coated Seahorse cell culture microplates and differentiated until day 100 as described above. On day 100, iPSC-neurons were incubated for 48 h with exosomes from Cv, R47H het R47H hom or TREM2 -/- iPSC-microglia. Data were analysed using Wave v2.4.0.6 software (Agilent Technologies) upon Crystal Violet normalization. 2.7. Cell stress array analysis iPSC-neurons were plated at a density of 50.000 cells/cm 2 , and on day 100, the cells were incubated with exosomes secreted from Cv, R47H hom , R47H het or TREM2 -/- microglia for 48 h. After treatment, cell lysates were prepared according to the manufacturer's instructions (Proteome ProfilesTM Human cell stress array; Bio-Techne). Total protein quantification was performed on the aliquots of each treatment group for data normalization purposes. Cell lysates from 3 independent neuronal inductions and exosomal extractions were pooled according to TREM2 genotype and exosomal treatments. The array blots were visualized using the Odyssey detection system (LiCor). Data were analysed and quantified using exported zip files in Image Studio Lite v5.2.d. 2.8. High-Content Assay for synaptic function To evaluate synaptic function and/or formation, iPSC-neurons were plated at a density of 50.000 cells/cm 2 in 96-well plates suitable for imaging with the Opera Phenix high-content screening system and cultured until day 100. Cells were then incubated with microglia exosomes, and after 24 h, PBS (Basal conditions) or amyloid-β (Aβ) 1-42 at 5 μM in PBS were also added to the media. Then, 48 h after Aβ 1-42 addition, cells were fixed with 4 % PFA in PBS followed by 0.1 % triton permeabilisation and blocking with 5 % normal goat serum. Cells were then incubated with primary antibodies SYN (1:500); Tuj1 (1:000); PSD95 (1:500); cleaved caspase-3 (1:500) at 4 o C overnight, followed by incubation with AlexaFluor 568 or 488 conjugated secondary antibodies (Supplementary table 1). Nuclei were counter-stained with DAPI. The iPSC-neuron plates were imaged with a 60X or 40X water-immersion objective using an Opera Phenix (PerkinElmer) high-content screening system. Automated image analysis was performed using Columbus software and an automated algorithm. The results are presented as the corrected mean fluorescent intensity (MFI) calculated as corrected sum fluorescent intensity normalized to total cell number per image region. Neurite parameters including their length (mean of total and max length) and the number of roots (The number of roots is the count of primary neurites (e.g., axons or dendrites) that emerge directly from the soma of a Tuj1-positive neuron) were determined with Tuj1 staining and automated algorithm in Columbus software. Measurements were performed in defined cell regions measured in 8-10 fields/well and different planes (z-stack) using 2-4 wells per condition (Supplementary table 1). 2. 9 . Cell viability assay To test whether exosomes were inducing cell death within the time course of the experiments (48h), iPSC-neurons were plated at a density of 50.000 cells per cm 2 in opaque tissue-culture-treated 96-well plates. On day 100 iPSC-neurons were incubated with microglia exosomes for 48 h, and Cell-Titer-Glo Luminescent Cell viability assay (Promega) was performed according to the manufacturer's protocol. 2.10. Glutamate release To evaluate the ability of exosomes to induce glutamate release by iPSC-neurons, cells were plated at a density of 50.000 cells per cm 2 until day 100, when they were incubated with microglial exosomes for 48 h in the presence or absence of Aβ 1-42 . Subsequently the supernatant was collected, centrifuged at 300g (av) for 15 min and the glutamate concentration was measured using a Glutamate Glo assay kit (Promega) according to the manufacturer’s instructions. 2.11. Statistical analysis The results were represented as mean of at least three independent experiments (three separate neuronal inductions; at least three independent exosomal extractions, from one to six iPSC-microglia lines per group) with r value of 0.05 or below considered significant. The results were analysed using Prism Software version 10. Analysis was performed on pooled control lines (2 control lines) and pooled R47H het lines (2 patient lines, 3 clones per patient line) 1 R47H hom line and 1 TREM2 -/- line with the isogenic line as one of the control lines. Data were analysed using unpaired t-test, Wilcoxon Signed Rank test, one- or two-way ANOVA followed by Tukey’s post hoc, or Kruskal-Wallis followed by Dunn’s post hoc, as indicated in figure legends. Data are presented at mean ± SEM. 3. Results 3.1. Exosomal isolation We successfully generated human cortical neurons from control iPSC, expressing at day 100 established neuronal markers including Tuj1, NeuN, TBR1, BRN2, SATB1 ( Figure 1a ). In parallel, we generated mature iPSC-microglia expressing TREM2 common variant (Cv), R47H het , R47H hom and TREM2 -/- ( Figure 1b ) from which we extracted exosomes (Cv exo, R47H het exo, R47H hom exo, TREM2 -/- exo, respectively). The exosomes presented with the expected size at the range of 200 nm and expressed the exosomal markers tested ( Figure 1c ) (as we also determined previously; Mallach et al., 2021). To explore the effect of Cv, R47H het , R47H hom TREM2 -/- iPSC-microglia secreted exosomes on neuronal gene expression profiles, we performed RNA-seq in iPSC-neurons incubated with microglia exosomes for 48 h. 3.2 Neuronal gene expression changes due to exosomal treatment from different TREM2 microglial lines Differential expression analyses showed altered expression of a number of genes including SULF1 , MMP2 , PLD1 between untreated neurons and neurons treated with TREM2 Cv exosomes ( Figure 2a ). These results were validated by qPCR where a panel of 6 genes were selected that showed significance in one of the comparisons shown throughout Figures 2 and 3: SULF1 , MMP2 , SSPO, IL33 and NPTX2 ( Figure 2b, Supplementary figure S1 ). The genes differentially expressed due to Cv microglial exosomes were significantly enriched for biological annotations associated with extracellular matrix and structure organization, cell migration and, glia cell derived neurotrophic factor (GDNF) receptor signalling pathway and Wingless-related integration site (Wnt) signalling pathway ( Figure 2c ). A higher number of genes showed increased expression in neurons treated with R47H het patient-derived microglial exosomes including 69 genes with altered expression in total (37 increased, 32 decreased) ( Figure 2d, Supplementary figure S1 ). Among them, the genes that were found increased were associated with metabolic pathways including cellular and mitochondrial fatty acid metabolism, and activity of related enzymes and channel activities. The genes with decreased expression were associated with growth factor complex ( Figure 2f ). Interestingly, SULF1 , MMP2 , IL33 and NPTX2 genes were found to be significantly increased by qPCR compared with control untreated neurons (Figure 2e). We found 53 genes differentially expressed in the neurons due to R47H hom -derived microglial exosomes with 29 upregulated and 24 downregulated ( Figure 2g, Supplementary figure S1 ). The upregulated genes were involved in synapse organization and density as well as pyruvate metabolic processes whereas the downregulated genes were associated with lipid metabolic processes and extracellular matrix organization ( Figure 2i ). Likewise, we found 24 genes differentially expressed in neurons following incubation with TREM2 -/- -derived microglial exosomes with 7 genes upregulated and 17 genes downregulated (Figure 2j, Supplementary figure S1). These genes were associated with lactate and pyruvate metabolic processes and matrix extracellular assembly (upregulated) and cerebellar atrophy, aggressive behavior and epileptic conditions (downregulated) (Figure 2l). SULF1 , MMP2, IL33, NPTX2, genes in TREM2 -/- exosome treated neurons were not significantly altered compared to control untreated neurons (Figure 2k). 3.3. Exosome induced neuronal gene changes due to microglial R47H expression or TREM2 loss. To explore alterations in a landscape more relevant to brain physiology, we performed differential expression analysis between Cv microglia exosome-treated neurons and R47H het patient (118 total genes: 67 upregulated, and 51 downregulated) or TREM2 -/- (90 total genes: 30 upregulated and 60 downregulated) microglia secreted exosomes. This analysis revealed a 2 to 5-fold increase in the number of differentially expressed genes in the selected conditions. More specifically 118 neuronal genes were shown to be differentially expressed due to exosomes derived from R47H het iPSC-microglia ( Figure 3a and b ). The genes upregulated in neurons were involved in neuronal ensheathment, myelin assembly and intracellular signalling pathways, whereas the downregulated neuronal genes were associated with nitrite-related processes, and cellular amino-acid metabolic processes ( Figure 3c ). Similarly, 90 neuronal genes were shown to be differentially expressed in neurons treated with exosomes secreted by microglia lacking TREM2 vs neurons treated with Cv microglial exosomes ( Figure 3d and e ). The genes upregulated in TREM2 -/- exosome-treated neurons were involved in mitochondrial respiration and cellular and oxidative phosphorylation, and pathways associated with neurodegeneration and AD, whereas the downregulated genes were involved in annotations associated with neurogenesis and neuronal differentiation and brain development ( Figure 3f ). 3.4. Exosomal effects on neuronal metabolism Given the observed impact of exosomes on the neuronal transcriptome related to metabolic processes, we explored the direct effect of the exosomes from TREM2 variants on neuronal oxidative phosphorylation (OXPHOS) by Seahorse analysis. Microglial exosomes did not significantly affect the spare respiratory capacity or ATP productions of the cells ( Figure 3g-i ); however, we observed a significant decrease in coupling efficiency and increased proton leak accordingly, in neurons incubated with exosomes secreted by microglia lacking TREM2 when compared with neurons treated with exosomes from Cv microglia ( Figure 3j-k ), confirming the transcriptomic results. 3.5. Exosomal effects on neurite outgrowth at basal and Aβ 1-42 induced damage To assess the effect of exosomes on the neurite network in our iPSC model we analysed the neuronal outgrowth. At basal conditions, iPSC-neurons treated with R47H het exosomes exhibited decreased neuronal outgrowth which did not reach significance when data from patient 1 (D, dementia) and 2 (nonD; non-dementia) were pooled. However, when we analysed separately data from patient 1 and 2, statistical analysis revealed a significant reduction of neurite outgrowth (max and total neurite length) and number of roots in neurons treated with exosomes from patient 1 (dementia patient) compared with untreated neurons ( Figure 4a and 4c-e ) at basal conditions, and did not protect against Aβ 1-42 -toxicity (Figure 4c and d). Likewise, neurons treated with TREM2 -/- exosomes presented a decreased neurite length at basal conditions and upon Aβ 1-42 treatment TREM2 -/- exosomes were not protective ( Figure 4d and e ). Aβ 1-42 treatment decreased neurite length, and interestingly, the addition of Cv exosomes, but not R47H het or TREM2 -/- derived exosomes showed a trend toward prevention, with a borderline statistical significance ( Figure 4a, c ). Similarly, the number of neurite roots was decreased in neurons treated with TREM2 variant derived exosomes compared to neurons treated with Cv exosomes upon Aβ stimulation ( Figure 4c ). 3.6. Exosomal effects on synaptic function and A β 1-42 related synaptic dysfunction To assess whether microglia exosomes deliver neuroprotective or detrimental effects at a synaptic level, we evaluated the expression of the presynaptic (SYN) and postsynaptic (PSD95) markers in our iPSC-neuron model. As expected Αβ 1-42 oligomers decreased significantly the expression of SYN ( Figure 4f ). Cv exosomes were shown to protect Αβ 1-42 challenged neurons in terms of their SYN expression, but R47H hom or TREM2 -/- ( Figure 4f ) or R47H het ( Figure 4g ) derived exosomes did not. Notably, R47H het from dementia patient 1 and TREM2 -/- microglial-secreted exosomes were shown to decrease basal levels of SYN expression ( Figure 4f-g ) which were not further decreased by Αβ 1-42 treatment. Microglial exosomes did not significantly alter PSD95 expression at basal ( Figure 4h ) or Aβ treated neurons. To test whether exosomal incubation affected glutamate release from neurons and whether TREM2 variant-derived exosomes altered this, we evaluated the levels of glutamate in neuronal supernatant after incubation with exosomes. Cv exosomes led to a significant increase in glutamate levels, and this increase was not detected following incubations with exosomes derived by TREM2 variant or TREM2 -/- microglia ( Figure 4i ), suggesting that these exosomes are not able to promote glutamate release and therefore a proper neuronal function. 3.7. Effect of exosomes on cell stress proteome and neuronal apoptosis Next, we assessed how TREM2 variant exosomes might affect the neuronal proteome with regard to cell stress ( Figure 5a-c ). Exosomes from Cv microglia led to an overall slight decrease in the expression of cell stress-related proteins in the neurons, such as Carbonic Anydrase IX, Cytochrome C, Hypoxia-Inducible factor -1α (HIF-1α), and Nuclear factor kappa B (NF-kB) ( Figure 5b and c ) and in turn, the expression of those showed a trend toward an increase upon incubation with R47H or TREM2 -/- microglia derived exosomes ( Figure 5b and c ). By evaluating the expression levels of cleaved caspase-3 we were able to determine whether TREM2 variant microglia exosomes promoted neuronal apoptosis. Our results showed an increasing trend in expression in neurons treated with TREM2 variant microglia exosomes, although it did not reach significance ( Figure 5e ). Upon an Αβ 1-42 challenge of 5 μM we did not observe any further induction of apoptosis, but consistently, an increasing trend of cleaved caspase-3 expression in neuronal groups treated with TREM2 variant exosomes was revealed ( Figure 5f ). To test whether exosomes affect neuronal viability at the concentration added, and thus, whether the observed changes at transcriptomic or functional (metabolic, synaptic or apoptotic) level were due to cell death we assessed the neuronal viability which was not altered within the 48 treatment window ( Figure 5g ). 4. Discussion Our data show that exposure of iPSC-derived cortical neurons to exosomes isolated from iPSC-microglia expressing common variant TREM2, the LOAD risk variant R47H het or the R47H hom and TREM -/- lines have significant and differing effects on the neuronal genome. In particular the genes affected are altered in different ways by the different line-derived exosomes. These findings extend our previous work in which we found that the exosomes themselves from different TREM2-variant iPSC-microglia show differential protein expression profiles and influenced SHSY5Y survival (Mallach et al., 2021a; b), again in a manner in which the TREM2 AD variant line-derived exosomes were not supportive of survival when compared with the Cv line-derived exosomes. Exosomal signaling (and that of larger extracellular vesicles, EVs) is becoming an increasingly important pathway in intercellular signaling (Hill, 2019; Wang and Xia, 2022). Exosomes are the most widely studied EVs and are derived from endosomes. We showed previously that microglial-derived exosomes can be taken up by neurons (Mallach et al., 2021a), suggesting this may be the route by which they can influence neuronal behaviour (Hooper et al., 2012). Interestingly, as with our findings for microglial-derived exosomes, oligodendrocytes can also release exosomes, which have been found to contain a number of proteins associated with protection against cell stress, and which has been argued could provide axonal support against injury (Krämer‐Albers et al., 2007). However to date, many of these studies have been performed on rodent derived exosomes (Hooper et al., 2012; Xie et al., 2022). These authors showed that astrocytes influenced neurodegenerative diseases through metabolic balance and ubiquitin-dependent protein balance, whereas microglial-derived exosomes influenced neurodegenerative diseases through immune inflammation and oxidative stress. Microglia exosomes have been shown to mediate neuroprotective or neurotoxic effects in a context dependent manner. In this study, healthy microglia exosomes were shown to primarily alter neuronal genes involved in extracellular matrix and structure organization, cell migration and motility. Extracellular matrix components which were upregulated by exosomal treatments such as SULF1 , MMP2 or COL1A1 have been reported to play a role in the formation, maintenance and function of synapses (Kalus et al., 2015; Condomitti and De Wit, 2018; Kamimura and Maeda, 2021) or associated with Aβ pathology and AD (Hosono-Fukao et al., 2012; Hernandez-Guillamon et al., 2015; Ozsan McMillan et al., 2023), further supporting a role of exosomes in neuronal support. TREM2 variant microglia derived exosomes also affected the expression of selected related genes when added to neurons, suggesting that this effect may be primarily mediated by changes in exosomal content due to TREM2 variants, and not directly related/altered due to TREM2. In line with the previously observed alterations in exosomal cargo related to metabolic processes due to R47H het (Mallach et al., 2021b), here, R47H het , R47H hom and TREM2 -/- microglia derived exosomes were shown to affect the human neuronal transcriptome associated with metabolic processes including lipid or lactate metabolism. Comparative analysis between R47H or TREM2 -/- and Cv exosome treated neurons further revealed differences in the expression of genes involved in various metabolic processes, including amino-acid metabolic pathways, oxidative phosphorylation and mitochondrial complexes confirming that the observed alterations can be attributed to TREM2 altered function or absence. Recently, it was shown that TREM2 -/- impairs neuronal transcriptome and bioenergetics in vivo (Tagliatti et al., 2024) and conditioned media from microglia expressing R47H decreased OXPHOS in human neurons (Vasilopoulou et al., 2024a). Thus, the present data suggest that these effects are mediated via mechanisms linked to exosomal pathways that are influenced by TREM2. Indeed, when TREM2 -/- exosomes were directly added to neurons, coupling efficiency and ATP production in neurons declined indicating mitochondrial dysfunction. In our experimental setting R47H exosomes did not significantly affect OCR parameters in real time metabolic analysis; however the observed trends follow the trend of TREM2 loss level (het>hom>absent). Interestingly, a set of genes altered via exosomes derived from TREM2 deficient microglia (compared with Cv exosomes) was associated with TCA cycle and respiratory electron transport. We previously showed that fueling microglia with citrate or succinate, two key TCA components, can rescue certain functional deficits due to R47H or TREM2 -/- and improve neuronal metabolic function via the secretome (Vasilopoulou et al., 2024a). This evidence suggests that exosomes, acting as key microglial secretome components, may be responsible for the observed effects, and additionally, ameliorating TREM2 deficient signalling in microglia and subsequently impaired metabolism may prevent negative exosome-mediated effects, due to TREM2 deficiency, on neuronal metabolism. Another set of differentially expressed genes pointed to alterations at synaptic processes and neuronal transmission delivered by microglial exosomes and influenced by TREM2 state, including axon and neuronal ensheathment, myelination, postsynaptic density and synapse organisation or neurogenesis. Previous research has shown an important role of microglia-derived EVs on synaptic plasticity and dysfunction (Gabrielli et al., 2022). These effects have been attributed in some cases on exosomal cargo of packaged miRNAs e.g. miR-223 enriched exosomes reduced neuroinflammation and ameliorated nerve damage in AD in vivo and in vitro (Wei et al., 2024). Among the differentially expressed genes neuronal NPTX2 was dysregulated in response to R47H derived exosomes. NPTX2 regulates complement activity and microglial synapse elimination in the brain (Zhou et al., 2023) and is considered a protective mechanism upon neuronal damage. However, recent findings have implicated NPTX2 in TDP-43-induced neurodegeneration, which characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in a human model of long-lived mature neurons (iPSC- colony morphology neural stem cells), suggesting a neurotoxic role under certain conditions (Hruska-Plochan et al., 2024). In this context, the neuronal dysregulation of NPTX2 may reflect a neuronal response following synaptic damage induced by exosomes. Indeed, post-translationally, exosomes derived from unstimulated variant microglia or microglia lacking TREM2 not only were less efficient at providing neuronal support but instead neurite outgrowth was decreased by LoF variant derived exosomes. Whether NPTX2 dysregulation in this setting contributes to a protective or a pathogenic response, as observed in ALS and FTD models deserves further exploration. Moreover, microglial exosomes promoted glutamate release which reflects a physiological neuronal function as previously shown (Antonucci et al., 2012) whereas exosomes lacking TREM2 did not. Consistently, Aβ-induced damage of the neural network was mitigated by Cv microglia exosomes but R47H or TREM2 -/- exosomes were unable to deliver any protective effects at such conditions. These data suggest a crucial role of TREM2 on neuronal health. Accordingly, at a synaptic level, Cv exosomes protected Aβ-induced synaptic damage but when TREM2 was deficient they did not. Interestingly, the synaptic effects of R47H exosomes were stronger when derived from R47H het carrier 1 who exhibited dementia in contrast to R47H het carrier 2 that did not manifest dementia symptoms, suggesting a potential link between the observed exosomal effect in vitro and cognitive state at a clinical level (Duggan et al., 2022) or reflecting background genomic variability between carriers. This observation aligns with GO enrichment annotations implicating TREM2 loss with diseases such as AD and PD. Whether the observed effects are due to the absence of “protective” cargo when TREM2 is deficient/absent or the presence of “toxic” cargo that is increased in exosomes released by TREM2 -/- microglia or both needs further exploration. It has been shown that under pathophysiological conditions exosomes can transfer pro-inflammatory damage associated molecular patterns (DAMPs) to surrounding cells. To further assess whether TREM2 LoF or absence compromises any beneficial/protective effects of microglial exosomes on neurons – or instead promotes detrimental effects, we investigated the cell stress-related neuronal proteome following microglial exosomal exposure. Overall, TREM2 loss was shown to prime cell stress related proteins (e.g HIF-1α, HIF-2α, Paraoxonases (PON) involved in metabolic/oxidative stress processes, which have been shown to be regulated by TREM2 in microglia cells (Ulland et al., 2017; March-Diaz et al., 2021). TREM2 R47H variant or TREM2 -/- exosomes seem to promote subtle apoptotic effects on neurons and further contribute to neuronal apoptosis when neurons are primed with Aβ 1-42 ; however, exosomal treatments do not affect cell viability by apoptosis over the time course of our experiments. We previously found that the R47H het -microglia display mitochondrial bioenergetic changes, with an inability to switch to glycolysis (Piers et al., 2020) and an inability to activate the inflammasome (Cosker et al., 2021). We show in the current work that neuronal oxidative phosphorylation is also altered by TREM2 LoF variants, which would likely impact on neuronal signalling responses. With regard to the inflammasome, a recent report suggests the inflammasome may facilitate exosome secretion of neurotoxic species such as a-synuclein in PD (Si et al., 2021). However whilst we cannot definitively say that a reduced inflammasome activity per se in R47H het iPSC-microglia directly influences exosome content (because we did not stimulate Cv or R47H microglia in a manner which would activate the inflammasome) (Cosker et al., 2021; Mallach et al., 2021b) we have shown that TREM2 LoF do produce an exosome population which is significantly different from Cv. It is also worth noting that the effects of the exosomes on neurons were assessed over a relatively short period of time. Even within that timeframe, we were able to detect a measurable degree of change. Future studies should aim to investigate the longer-term consequences of microglial exosomal exposure on neuronal function and viability. Cellular functional consequences due to TREM2 impairment in microglia extend to neurons, but the precise intercellular mechanisms are not determined. Here, we show that TREM2 can shape the effects of human microglial exosomes on neuronal transcriptome profile and functions. Our data provide insights into the intercellular exosomal pathways whereby microglia affect neuronal heath in a TREM2-dependent manner. Taken together, our data suggest that alongside the direct cell-to-cell contact and the soluble factor release, microglia can communicate with neurons by bidirectional release of exosomes. Microglial exosomes contain TREM2 as part of their cargo (Mallach et al., 2021b; Huang et al., 2022) suggesting a role of TREM2 on subsequent exosomal mediated effects. There is likely to be further bi-directional cross talk and increased complexity following the involvement of oligodendrocytes and astrocytes in these studies. Abbreviations AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; bFGF, basic fibroblast growth factor; EVs, Extracellular vesicles; FTD, frontotemporal dementia; GDNF, glia cell derived neurotrophic factor; iPSC, induced pluripotent stem cell; LOAD, Late-onset Alzheimer’s disease; LoF, loss-of-function; NIM, neuronal induction media; NMM, neuronal maintenance media; NPC, neuronal progenitor cells; PLO, poly-L-ornithine; TREM2, triggering receptor expressed on myeloid cells-2; Wnt, Wingless-related integration site Declarations Funding statement: We acknowledge the Rosetrees Trust, UK for providing funding for the salary of FV and research costs of this project, (Funding to JMP, SW and DS). We acknowledge support for DS from the Dolby Foundation, USA (to JH) and the Dementia Research Institute, University College London (to JH). SW and JH are supported by the UCLH NIHR Biomedical Research Centre. 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Additional Declarations No competing interests reported. Supplementary Files SupplementarytablesRNAseq38.xlsx Supplementarytables12.docx SupplementaryFigureS1neditV3.pdf Supplementary Figure 1. Heatmaps showing differentially expressed genes for neurons treated with (a,b) R47H het or (d,e) TREM2 -/- exosomes versus neurons treated with a) Cv, (d) R47H het , (g) R47H hom or (i) TREM2 -/- exosomes versus non-treated (Control) neurons. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7487794","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513379792,"identity":"97038923-64b8-4063-bb12-cdb0cae2d107","order_by":0,"name":"Foteini Vasilopoulou","email":"","orcid":"","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Foteini","middleName":"","lastName":"Vasilopoulou","suffix":""},{"id":513379793,"identity":"fe392b8f-dd9d-43c4-ae5e-92f389dee034","order_by":1,"name":"Umran Yaman","email":"","orcid":"","institution":"UK Dementia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Umran","middleName":"","lastName":"Yaman","suffix":""},{"id":513379794,"identity":"2ae17ab8-c536-469f-88f9-8cb6c61743f6","order_by":2,"name":"Dervis A. Salih","email":"","orcid":"","institution":"UK Dementia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Dervis","middleName":"A.","lastName":"Salih","suffix":""},{"id":513379795,"identity":"905f9dee-987b-4369-a51f-86f905cb554b","order_by":3,"name":"John Hardy","email":"","orcid":"","institution":"UK Dementia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Hardy","suffix":""},{"id":513379796,"identity":"ab60baa8-325b-4ea5-b58e-0309724e1533","order_by":4,"name":"Selina Wray","email":"","orcid":"","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Selina","middleName":"","lastName":"Wray","suffix":""},{"id":513379797,"identity":"2c4f8193-f332-4c60-8bcd-0e363383a845","order_by":5,"name":"Jennifer M Pocock","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBACxgYIbcAPZB8AM3mI1SIJZBCnBQYMDA4Qq4W5gffggx8VdsbGN5IfHGCosWMwOHOAkMP4kg17ziSbmd1IA1p0LJnB4GwDIS08ZtKMbQdszG7kAB3GdoDB4DwBh8G1GM8AaflHghYzAwmgFiCDCIc1Q/xiLHHmmcGBxL5kHklC3jds7wWHmGF/e/LDBx++2cnxnUkgoKUZORoSiIlIeaIjexSMglEwCkYuAADgkkDvRyuhWwAAAABJRU5ErkJggg==","orcid":"","institution":"University College London","correspondingAuthor":true,"prefix":"","firstName":"Jennifer","middleName":"M","lastName":"Pocock","suffix":""}],"badges":[],"createdAt":"2025-08-29 10:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7487794/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7487794/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91537289,"identity":"8da22373-2aac-4e2a-8b2a-e15518873031","added_by":"auto","created_at":"2025-09-17 13:14:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1568193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterisation of iPSC-neurons, microglia and microglial-derived exosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Cell culture images at critical points of the neuronal differentiation and characterization by immunocytochemistry (ICC) staining for neuronal markers including Tuj1, NeuN, TBR1 (Layer VI;deep layer), BRN2 and SATB2 (Layer I,II; upper layer). (b) Cell culture images at critical points of the microglial differentiation and characterization by ICC staining for microglia marker IBA-1. (c) i) Transmission electron microscopy of iPSC-microglia exosomes showing expected size range of 30-200nm and ii) representative blots for exosomal markers (ALIX, HSP70) and β-actin. (d) Schematical representation of experimental design followed in this study.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/9f89c72142f8ae71db8aba21.png"},{"id":91538584,"identity":"3fe8b5ea-6ae5-48d8-8f31-dc2a809f93c6","added_by":"auto","created_at":"2025-09-17 13:30:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":142466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression profiles in iPSC-neurons treated with microglial exosomes \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVolcano plot showing differentially RNAseq analysis of expressed genes for neurons treated with (a) Cv, (d) R47H\u003csup\u003ehet\u003c/sup\u003e, (g) R47H\u003csup\u003ehom\u003c/sup\u003e or (i) TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes versus non-treated (Control) neurons. Gene expression levels of selected genes (\u003cem\u003eSULF1\u003c/em\u003e, \u003cem\u003eMMP2, IL33, SSPO, NPTX2\u003c/em\u003e) by qPCR in neurons treated with (b) Cv, (e) R47H\u003csup\u003ehet\u003c/sup\u003e, (h) R47H\u003csup\u003ehom\u003c/sup\u003e or (k) TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes versus non-treated (Control) neurons. Data are presented as mean ± SEM. Statistical significance was addressed with Wilcoxon Signed Rank Test. Biological annotation associated with differentially expressed genes for neurons treated with (c) Cv, (f) R47H\u003csup\u003ehet\u003c/sup\u003e, (i) R47H\u003csup\u003ehom\u003c/sup\u003e or (l) TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes versus non-treated (Control) neurons. *ρ\u0026lt;0.05; (N\u003csub\u003etotal\u003c/sub\u003e=3-11; independent neuronal inductions (n=3); microglia derived exosomal samples per group (n≥3).\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/2b5c78f4cc33920fc9a1657a.png"},{"id":91537643,"identity":"a1e4ffef-e7d6-48ab-8153-418342452fc0","added_by":"auto","created_at":"2025-09-17 13:22:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":93756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression profile in iPSC-neurons treated with microglial exosomes from R47H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehet\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e or TREM2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells compared to Cv and effects on neuronal metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeatmaps and volcano plots showing differentially expressed genes for neurons treated with (\u003cstrong\u003ea,b\u003c/strong\u003e) R47H\u003csup\u003ehet\u003c/sup\u003e or \u003cstrong\u003e(d,e)\u003c/strong\u003e TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes versus neurons treated with Cv exosomes. Biological annotation associated with differentially expressed genes between neurons treated with \u003cstrong\u003e(c)\u003c/strong\u003e R47H\u003csup\u003ehet\u003c/sup\u003e or Cv exosomes and \u003cstrong\u003e(f)\u003c/strong\u003e TREM2\u003csup\u003e-/-\u003c/sup\u003e or Cv exosomes. \u003cstrong\u003e(g)\u003c/strong\u003e Real time OCR for neurons treated with microglial exosomes, (\u003cstrong\u003eh\u003c/strong\u003e) spare respiratory capacity (\u003cstrong\u003ei\u003c/strong\u003e) ATP production \u003cstrong\u003e(j)\u003c/strong\u003e coupling efficiency (\u003cstrong\u003ek\u003c/strong\u003e) proton leak in iPSC-neurons treated with exosomes secreted by Cv, R47H\u003csup\u003ehet\u003c/sup\u003e, R47H\u003csup\u003ehom\u003c/sup\u003e or TREM2\u003csup\u003e-/-\u003c/sup\u003e iPSC-microglia. Data are presented as mean ± SEM. Statistical significance was addressed with one or two-way ANOVA followed by Tukey’s post-hoc. *ρ\u0026lt;0.05; **ρ\u0026lt;0.01; (N\u003csub\u003etotal\u003c/sub\u003e=5-27; independent neuronal inductions (n=3); microglia derived exosomal samples per group (n≥3).\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/edd80e5ac0971ff5cf713edc.png"},{"id":91537294,"identity":"e6d53571-851a-4f46-b59f-7dbd55a69f94","added_by":"auto","created_at":"2025-09-17 13:14:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1816520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and functional changes in iPSC-neurons exposed to microglial exosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Representative images for synaptophysin (SYN) and Tuj1 imaged by Opera Phenix and quantification of \u003cstrong\u003e(c)\u003c/strong\u003e neurite number of roots \u003cstrong\u003e(d)\u003c/strong\u003e neurite mean total length \u003cstrong\u003e(e) \u003c/strong\u003eneurite mean max length, \u003cstrong\u003e(f-g)\u003c/strong\u003e SYN expression in iPSC-neurons treated with microglial exosomes at basal conditions or after incubation with Aβ\u003csub\u003e1-42\u003c/sub\u003e at 5μM. \u003cstrong\u003e(b)\u003c/strong\u003e Representative images for PSD95 and \u003cstrong\u003e(h) \u003c/strong\u003equantification in iPSC-neurons treated with microglial exosomes at basal conditions after incubation with Aβ\u003csub\u003e1-42\u003c/sub\u003e at 5μM. \u003cstrong\u003e(i)\u003c/strong\u003e Glutamate release in neuronal supernatant after incubations with microglial exosomes. Data are presented as mean ± SEM. Statistical significance was addressed with one or two-way ANOVA followed by Tukey’s post-hoc. *ρ\u0026lt;0.05; **ρ\u0026lt;0.01; ***ρ\u0026lt;0.001; (N\u003csub\u003etotal\u003c/sub\u003e=2-11; independent neuronal inductions (n=3); microglia derived exosomal samples per group (n≥2).\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/9e89e6d0aa7c1e05f1610a87.png"},{"id":91537288,"identity":"b1f53bc8-9141-46f0-9f1e-9d91de9d4322","added_by":"auto","created_at":"2025-09-17 13:14:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1136812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of exosomes on neuronal stress proteome and survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative array blots and (b) heatmaps showing expression levels of neuronal stress-related proteins in the presence or absence of microglia exosomes. (c) Bar chart showing expression of stress related proteins fold-change to non-treated (Control) iPSC-neurons levels. (d) Representative images for cleaved-caspase 3 and Tuj1 imaged by Opera Phenix and quantification of (e) levels at basal conditions or (f) upon Aβ\u003csub\u003e1-42 \u003c/sub\u003estimulation. (g) Cell viability as assessed with Cell-Titer Glo viability assay. Data are presented as mean ± SEM Statistical significance was addressed with one or two-way ANOVA followed by Tukey’s post-hoc. *ρ\u0026lt;0.05; (N\u003csub\u003etotal\u003c/sub\u003e =3-24 independent neuronal inductions (n=3); microglia derived exosomal samples per group (n≥3).\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/c0ecc81f9fe57a831e4d3c31.png"},{"id":92628490,"identity":"b2c19e2f-0189-47d2-8fa6-87e514fb55fa","added_by":"auto","created_at":"2025-10-02 01:46:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6089462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/6000792c-356f-4ee3-ae47-e847db6d7080.pdf"},{"id":91537645,"identity":"2919c033-709c-4eb4-87aa-2f2bf043c171","added_by":"auto","created_at":"2025-09-17 13:22:21","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49810,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytablesRNAseq38.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/0f57d326e0c4e53fdec0aad6.xlsx"},{"id":91537285,"identity":"f0191ae7-40d4-4ba3-b491-d833452177d0","added_by":"auto","created_at":"2025-09-17 13:14:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21699,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables12.docx","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/6423f35bef0d44e3e28f6b24.docx"},{"id":91537648,"identity":"08db6425-2970-4811-aa27-42296f97175e","added_by":"auto","created_at":"2025-09-17 13:22:21","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":163881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. \u003c/strong\u003eHeatmaps showing differentially expressed genes for neurons treated with (\u003cstrong\u003ea,b\u003c/strong\u003e) R47H\u003csup\u003ehet\u003c/sup\u003e or \u003cstrong\u003e(d,e)\u003c/strong\u003e TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes versus neurons treated with a) Cv, (d) R47H\u003csup\u003ehet\u003c/sup\u003e, (g) R47H\u003csup\u003ehom\u003c/sup\u003e or (i) TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes versus non-treated (Control) neurons.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS1neditV3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7487794/v1/1b6dac2623013667b7c2bdd0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of iPSC-derived microglial exosomes on neurons: Role of TREM2 and implication in Alzheimer's Disease","fulltext":[{"header":"Main points","content":"\u003cul\u003e\n \u003cli\u003eExosomes secreted by the R47H variant or TREM2\u003csup\u003e-/-\u003c/sup\u003e iPSC-microglia differentially regulate the neuronal transcriptome.\u003c/li\u003e\n \u003cli\u003eTREM2 play a role in modulating neuronal metabolism, neurite outgrowth and synaptic function via exosomal pathways.\u003c/li\u003e\n \u003cli\u003eR47H and TREM2\u003csup\u003e-/-\u003c/sup\u003e microglia-derived exosomes fail to \u003cstrong\u003eprotect neurons from synaptic dysfunction\u003c/strong\u003e upon A\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e challenge\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eMicroglia, the immune cells of the brain, interact with nearly all brain cell types to support developmental processes, maintain homeostasis, contribute to tissue repair, and play a role in the pathogenesis of diseases (Gao et al., 2023). Microglial triggering receptor expressed on myeloid cells 2 (TREM2) regulates microglial functions including phagocytosis, metabolism, immune activation and recent evidence suggests that TREM2 plays a crucial role in microglia-neuron cross talk (reviewed in (Pocock et al., 2024). Rare TREM2 variants, including the loss-of-function (LoF) R47H variant, have been associated with an increased risk of late-onset Alzheimer\u0026rsquo;s Disease (AD), the most prevalent form of AD. Microglia expressing LoF TREM2 variants or those completely lacking TREM2 exhibit phagocytic, metabolic, immune activation deficits and affect neuronal health (Garcia-Reitboeck et al., 2018; Piers et al., 2020; Cosker et al., 2021; Popescu et al., 2023; Tagliatti et al., 2024). However, the pathways whereby TREM2-dependent microglia shape neuronal function, and how AD risk variants impact upon these pathways remain largely underexplored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExtracellular vesicles (EVs) are shed from cells and are nano-sized particles which can be classified broadly into exosomes (40-150 nm), microvesicles (50-2000 nm), or apoptotic bodies (50-5000 nm). Exosomes are small EVs, ranging from 40-200 nm in size originating from the endocytic pathway and carrying a cargo of nucleic acids, proteins, and lipids that vary, reflecting their cellular origin and state (Antonucci et al., 2012; Gupta and Pulliam, 2014; Yang et al., 2018; Mathieu et al., 2019). Exosomes are key microglia secretome components and once released are taken into recipient cells, where they facilitate intracellular communication and crosstalk with other brain cells, or even propagate disease states (Guo et al., 2021; Vasilopoulou et al., 2024b). We have previously demonstrated that the TREM2 LoF R47H variant results in an altered content of the exosomes released by human induced pluripotent stem cell (iPSC) - microglia, particularly with regard to metabolic and transcription pathway changes (Mallach et al., 2021a; b). At the same time, TREM2 has been shown to affect exosomal-mediated spreading and the seeding of pathological AD associated proteins (Leyns et al., 2019; Zhu et al., 2022). This evidence suggests that TREM2, which can be found in the exosomal content and membrane of the exosomes (Mallach et al., 2021a; b) can influence microglial intercellular communication pathways, affecting various pathological events and thereby contributing to disease progression. In this study we aimed to go deeper into the TREM2-dependent beneficial, or detrimental effects delivered by exosomes secreted by human iPSC-microglia harbouring the LoF R47H variant or lacking TREM2, through transcriptomic, proteomic and functional assessment of neurons. We hypothesise that TREM2 variant microglial exosomes will lead to changes to neuronal transcriptome translated into functional changes that may promote beneficial or detrimental neuronal processes in a TREM2-dependent manner. Our approach provides insights into the disease mechanisms underlying microglial-driven neurodegenerative processes in AD and other diseases characterized by neuronal dysfunction.\u003c/p\u003e"},{"header":"2.\tMaterials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 iPSC-neuronal culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe control iPSC line BIONi010-C (EBiSC) was used for the experiments. iPSCs were differentiated into cortical neurons as described in \u003cu\u003eShi et al., (2012\u003c/u\u003e) with slight modifications. Briefly, iPSC were grown to 100% confluence in E8 media, and neural induction started (Day 0) by switching media to neuronal induction media (NIM), which consisted of neuronal maintenance media (NMM) supplemented with 10\u0026mu;M SB-431542 (Sigma) and 200nM LDN-193189 (Sigma). NIM was replaced daily for 10-12 days until a homogeneous neuroepithelial sheet was formed. On day 12, neuroepithelial cells were passaged using dispase to laminin-coated plates (50\u0026mu;g/mL, Gibco), and the medium was switched to NMM (which consisted of 1:1 mixture of Dulbecco\u0026apos;s modified eagle medium F12 (DMEM-F12) and Neurobasal supplemented with 0.5x B27, 0.5x N2, 1.7x GlutaMAX, 0.5x sodium pyruvate, 25\u0026mu;g/ml Pen/Strep, 50\u0026mu;M 2-mercaptoethanol, 0.5x nonessential amino acids, 2.5 \u0026mu;g/ml insulin. For the next 3 days, 20ng/ml bFGF was added to NMM to promote rosette expansion. On day 16, bFGF was withdrawn, and cells were maintained to NMM until day 25, changing the media every other day. After expansion, around days 23-25, neuronal progenitor cells (NPC) were passaged with Accutase (Sigma) to laminin-coated plates \u003cstrong\u003e(Figure 1a).\u003c/strong\u003e NMM was replaced every other day, and NPC were passaged every 2-4 days using Accutase until clear neuronal projections were formed. On day 33 after induction cells were dissociated with Accutase for the final time and plated in laminin (50\u0026mu;g/mL, Gibco) /poly-L-ornithine (PLO) (0.01% wt/vol, Sigma) coated plates, at a final density of 50,000 cells per cm\u003csup\u003e2\u003c/sup\u003e. When cells completed 95-100 days in culture, mature human iPSC-neurons were used for the experiments. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo characterize cortical neuron cultures, iPSC-neurons were seeded and matured on 13 mm glass coverslips. On day 100, the cells were fixed with 4%PFA in Dulbecco\u0026apos;s phosphate buffered saline (PBS) for 20 min at room temperature (RT) following permeabilisation with 0.1% Triton X. Unspecific binding sites were blocked with 5% normal goat serum for 1h at RT, and the cells were incubated with primary antibodies for staining of established neuronal markers, including Tuj1 (1:1000), NeuN (1:100), TBR1 (1:500) BRN2 (1:500), SATB2 (1:200), synaptophysin (1:500) at 4\u003csup\u003eo\u003c/sup\u003eC overnight.\u0026nbsp;The cells were then incubated with anti-mouse or anti-rabbit AlexaFluor 488 or 568 conjugated secondary antibodies (1:500) and nuclei were counterstained with DAPI. Different regions per coverslip were imaged on a Zeiss LSM710 confocal microscope (40X, 63X) using the LSM Pascal 5.0 software. Brightfield images were obtained at critical points of the neuronal differentiation using a Leica microscope (10X, 20X) (\u003cstrong\u003eFigure 1a)\u003c/strong\u003e. All antibodies used are listed in \u003cstrong\u003esupplementary table S1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 iPSC-derived microglia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo control iPSC lines were used: SFC840 (Stembancc), and BIONi010-C (EBiSC), to generate \u003cem\u003eTREM2\u003c/em\u003e common variant (Cv) microglia. R47H heterozygous fibroblasts were previously acquired with an MTA between University College London and University of California Irvine Alzheimer\u0026apos;s Disease Research Center (UCI ADRC; Prof M Blurton-Jones) and fibroblast reprogramming and Karyotyping of a number of lines and clones described previously (Piers et al., 2020). Three clones from two ADRC R47H heterozygous (R47H\u003csup\u003ehet\u003c/sup\u003e) patient lines were used: ADRC8 (clones 3, 6, and 12), and ADRC26 (clones 3, 5, and 15). TREM2 R47H homozygous (R47H\u003csup\u003ehom\u003c/sup\u003e; BIONi010-C7) and TREM2 knock-out (TREM2\u003csup\u003e-/-\u003c/sup\u003e; BIONi010-C17) were gene edited BioNi010-C, all purchased from EBiSC. All iPSC were maintained and routinely passaged in Essential 8 medium. iPSC-microglia were generated using our previously described protocol (Piers et al., 2020) which incorporates procedures from earlier protocols (Garcia-Reitboeck et al., 2018; Xiang et al., 2018).\u0026nbsp;Briefly, embryoid bodies were generated from 70% confluent iPSC and maintained for 5 days in 100 ng/ml ROCK inhibitor, 50 ng/ml VEGF, 50 ng/ml BMP-4 and 20 ng/ml SCF. The embryoid bodies were then transferred into flasks where they were maintained in X-VIVO (Lonza) supplemented with 100 ng/ml MCSF and 25 ng/ml IL3 for further differentiation. After 4-5 weeks, progenitor cells were collected and plated in iPSC-microglia\u0026nbsp;maintenance medium supplemented with 100 ng/ml IL-34, 25 ng/ml MCSF, and 5 ng/ml TGF-\u0026beta;.\u0026nbsp;Medium was changed every week and 2 weeks after plating, 100 ng/ml CX3CL1 and 100 ng/ml CD200\u0026nbsp;were added to microglia medium (maturation medium) and subsequently to the microglia for 3 days to achieve final maturation (\u003cstrong\u003eFigure 1b).\u003c/strong\u003e All growth factors were purchased from PeproTech (Thermo Fisher Scientific).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3\u003c/strong\u003e. \u003cstrong\u003eExosome collection and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003echaracterisation by Transmission Electron microscopy and Western Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe iPSC-microglia were plated and matured on 6-well plates at a density of 50.000 cells/cm\u003csup\u003e2\u003c/sup\u003e. Medium was changed on the iPSC-microglia 48 h before the experiment. The supernatant was collected, centrifuged for 15 min at 300g\u003csub\u003e(av)\u003c/sub\u003e and subsequently used for exosome collection. Exosomes were extracted using an ExoQuick-TC kit (System Biosciences) by centrifugation of the supernatant for 15 min at 3000g\u003csub\u003e(av)\u003c/sub\u003e\u0026nbsp; followed by ExoQuick solution addition overnight at 4\u003csup\u003e\u0026omicron;\u003c/sup\u003eC. After two subsequent centrifugations at 1500g\u003csub\u003e(av)\u003c/sub\u003e\u0026nbsp; for 30 min and 5 min, the pellets were collected and resuspended in PBS without Ca\u003csup\u003e2+/\u003c/sup\u003eMg\u003csup\u003e2+\u003c/sup\u003e, pH 7.0\u0026ndash;7.3 or RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Exosomal content was quantified by performing Micro BCA\u003csup\u003eTM\u003c/sup\u003e protein quantification (Invitrogen, Thermo Fisher Scientific) and used for neuronal treatments at a concentration of 1\u0026nbsp;\u0026mu;g/10,000 cells as described previously\u0026nbsp;(Mallach et al., 2021a; b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExosome samples for transmission electron microscopy (TEM) were dropped with a Pasteur pipette onto a carbon/formvar coated copper grid. After 15 seconds excess sample was blotted off with filter paper. Then a drop of 2% phosphotungstic acid stain was added and blotted after 15 seconds. The grid was placed into a specimen holder and inserted into a Phillips/FEI CM 120 BioTwin TEM for imaging at 80kV \u003cstrong\u003e(Figure 1ci)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExosomal marker expression was confirmed by western blot. Briefly, exosomal pellets or iPSC-microglia cells were lysed in RIPA buffer and centrifuged at 15,000g\u003csub\u003e(av)\u003c/sub\u003e\u0026nbsp; for 15 min and the samples denatured and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and blocked with 5% milk in Tris buffered saline solution with 1% Tween -20 (TBS-T), and incubated with primary antibodies including ALIX (1:000), HSP70 (1:1000) and \u0026beta;-actin (1:5000) (Supplementary table S1) overnight at 4\u003csup\u003eo\u003c/sup\u003eC followed by incubation with the appropriate HRP-conjugated secondary antibody at 1:5000 for 1h at RT\u003cstrong\u003e\u0026nbsp;(Figure 1cii).\u0026nbsp;\u003c/strong\u003eThe membranes were washed 3x with TBS-T followed by a final TBS-T wash and visualized using the Odyssey detection system (LiCor).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. RNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eiPSC-neurons were plated at a density of 50,000 cells/cm\u003csup\u003e2\u003c/sup\u003e, and on day 100 the cells were incubated with iPSC-microglia exosomes for 48 h. After treatment, iPSC-neurons were lysed in RNA protection buffer (New England Biolabs) and used for RNA extraction following the manufacturer\u0026rsquo;s instructions (Monach Total RNA, New England Biolabs). RNA concentration and 260/230 260/280 ratios were measured by NanoDrop (DeNovix DS-11FX+spectophotometer). Quality of the total RNA was assessed using capillary electrophoresis.The RNA-seq library preparation and sequencing was performed by Novogene, UK. Eukaryotic RNA-seq libraries were prepared using the NEBNext\u0026reg; Ultra\u0026trade; RNA Library preparation Kit, generating 250-300 bp cDNA inserts. Quantified libraries were pooled and sequenced by Illumina NovaSeq6000 generating 150-bp paired-end (PE150) reads, multiplex samples per lane - 9G data per sample. From FASTQ files, adaptors and low quality base pairs were removed. Transcripts were aligned with HISAT2 (Mortazavi et al., 2008), using gene annotation from human reference genome GRCh38/hg38. RNA-seq count data were normalized using the DESeqDataSetFromMatrix() function in DESeq2 (Love et al., 2014), with default parameters and a design formula ~ Batch + Genotype of origin of exosomes to model batch as a covariate during differential expression analysis. DESeq2 uses raw read counts, applies normalization, and estimates dispersion. The effect of genotype of the origin of exosomes was tested, and differentially expressed genes were stated as r value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and fold-change \u0026sup3; 0.5. For visualization using heatmaps, variance-stabilizing transformation was applied using vst(), and batch effects were further removed from the transformed data using limma::removeBatchEffect() with default settings. Biological annotations were identified against Gene Ontology, REACTOME (Fabregat et al., 2018) and KEGG databases using gProfileR2 (Kanehisa et al., 2016; Raudvere et al., 2019), using genes with r value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and fold-change \u0026sup3; 0.5, using all detected genes in our experiment as the background.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Real-time qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative qPCR experiments were performed to determine the effect of exosomes on iPSC-neurons and for RNA-seq validation. RNA was extracted as described in section \u003cstrong\u003e2.4\u003c/strong\u003e. cDNA was generated with a high-capacity cDNA reverse transcription kit (Applied Biosystems) and qPCR analysis was performed using Taqman Universal Master Mix (Life Technologies) using specific primers (Supplementary table 2) and the MxPro qPCR software (Agilent). Expression was normalized to GAPDH. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Seahorse Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor real-time analysis of oxygen consumption rates (OCR) we used the Seahorse XF Cell Mito Stress Test kit (Agilent Technologies) as previously described for our iPSC-microglia (Garcia-Reitboeck et al., 2018; Cosker et al., 2021; Vasilopoulou et al., 2024a).\u0026nbsp;iPSC-neurons were plated at a density of 50.000\u0026nbsp;per cm\u003csup\u003e2\u003c/sup\u003e,\u0026nbsp;on\u0026nbsp;laminin/PLO coated\u0026nbsp;Seahorse cell culture microplates and differentiated until day 100 as described above. On day 100, iPSC-neurons were incubated for 48 h with exosomes from Cv, R47H\u003csup\u003ehet\u003c/sup\u003e R47H\u003csup\u003ehom\u003c/sup\u003e or TREM2\u003csup\u003e-/-\u003c/sup\u003e iPSC-microglia. Data were analysed using Wave v2.4.0.6 software (Agilent Technologies) upon Crystal Violet normalization. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Cell stress array analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eiPSC-neurons were plated at a density of 50.000\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells/cm\u003csup\u003e2\u003c/sup\u003e, and on day 100, the cells were incubated with exosomes secreted from Cv, R47H\u003csup\u003ehom\u003c/sup\u003e, R47H\u003csup\u003ehet\u003c/sup\u003e or TREM2\u003csup\u003e-/-\u003c/sup\u003e microglia for 48 h. After treatment, cell lysates were prepared according to the manufacturer\u0026apos;s instructions (Proteome ProfilesTM Human cell stress array; Bio-Techne). Total protein quantification was performed on the aliquots of each treatment group for data normalization purposes. Cell lysates from 3 independent neuronal inductions and exosomal extractions were pooled according to TREM2 genotype and exosomal treatments. The array blots were visualized using the Odyssey detection system (LiCor). Data were analysed and quantified using exported zip files in Image Studio Lite v5.2.d. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8.\u003c/strong\u003e \u003cstrong\u003eHigh-Content Assay for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esynaptic function\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate synaptic function and/or formation, iPSC-neurons were plated at a density of\u0026nbsp;50.000\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells/cm\u003csup\u003e2\u0026nbsp;\u003c/sup\u003ein 96-well plates suitable for imaging with the Opera Phenix high-content screening system and cultured until day 100. Cells were then incubated with microglia exosomes, and after 24 h, PBS (Basal conditions) or amyloid-\u0026beta; (A\u0026beta;) \u003csub\u003e1-42\u003c/sub\u003e at 5 \u0026mu;M in PBS were also added to the media. Then, 48 h after A\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e addition, cells were fixed with 4 % PFA in PBS followed by 0.1 % triton permeabilisation and blocking with 5 % normal goat serum. Cells were then incubated with primary antibodies SYN (1:500); Tuj1 (1:000); PSD95 (1:500); cleaved caspase-3 (1:500) at 4\u003csup\u003eo\u003c/sup\u003eC overnight, followed by incubation with AlexaFluor 568 or 488 conjugated secondary antibodies (Supplementary table 1). Nuclei were counter-stained with DAPI. The iPSC-neuron plates were imaged with a 60X or 40X water-immersion objective using an Opera Phenix (PerkinElmer) high-content screening system. Automated image analysis was performed using Columbus software and an automated algorithm. The results are presented as the corrected mean fluorescent intensity (MFI) calculated as corrected sum fluorescent intensity normalized to total cell number per image region. Neurite parameters including their length (mean of total and max length) and the number of roots (The number of roots is the count of primary neurites (e.g., axons or dendrites) that emerge directly from the soma of a Tuj1-positive neuron) were determined with Tuj1 staining and automated algorithm in Columbus software. Measurements were performed in defined cell regions measured in 8-10 fields/well and different planes (z-stack) using 2-4 wells per condition (Supplementary table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003cstrong\u003e. Cell viability assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether exosomes were inducing cell death within the time course of the experiments (48h), iPSC-neurons were plated at a density of 50.000\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells per cm\u003csup\u003e2\u003c/sup\u003e in opaque tissue-culture-treated 96-well plates. On day 100 iPSC-neurons were incubated with microglia exosomes for 48 h, and Cell-Titer-Glo Luminescent Cell viability assay (Promega) was performed according to the manufacturer\u0026apos;s protocol. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10. Glutamate release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the ability of exosomes to induce glutamate release by iPSC-neurons, cells were plated at a density of 50.000\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecells per cm\u003csup\u003e2\u003c/sup\u003e until day 100, when they were incubated with microglial exosomes for 48 h in the presence or absence of A\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e. \u0026nbsp;Subsequently the supernatant was collected, centrifuged at 300g\u003csub\u003e(av)\u003c/sub\u003e\u0026nbsp; \u0026nbsp;for 15 min and the glutamate concentration was measured using a Glutamate Glo assay kit (Promega) according to the manufacturer\u0026rsquo;s instructions. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results were represented as mean of at least three independent experiments (three separate neuronal inductions; at least three independent exosomal extractions, from one to six iPSC-microglia lines per group) with\u0026nbsp;r\u0026nbsp;value of 0.05 or below considered significant. The results were analysed using Prism Software version 10. Analysis was performed on pooled control lines (2 control lines) and pooled R47H\u003csup\u003ehet\u0026nbsp;\u003c/sup\u003elines (2 patient lines, 3 clones per patient line) 1 R47H\u003csup\u003ehom\u003c/sup\u003e line and 1 TREM2 \u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003eline with the isogenic line as one of the control lines. Data were analysed using unpaired t-test, Wilcoxon Signed Rank test, one- or two-way ANOVA followed by Tukey\u0026rsquo;s post hoc, or Kruskal-Wallis followed by Dunn\u0026rsquo;s post hoc, as indicated in figure legends. Data are presented at mean \u0026plusmn; SEM.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Exosomal isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe successfully generated human cortical neurons from control iPSC, expressing at day 100 established neuronal markers including Tuj1, NeuN, TBR1, BRN2, SATB1 (\u003cstrong\u003eFigure 1a\u003c/strong\u003e). In parallel, we generated mature iPSC-microglia expressing TREM2 common variant (Cv), R47H\u003csup\u003ehet\u003c/sup\u003e, R47H\u003csup\u003ehom\u0026nbsp;\u003c/sup\u003eand TREM2\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e(\u003cstrong\u003eFigure 1b\u003c/strong\u003e) from which we extracted exosomes (Cv exo, R47H\u003csup\u003ehet\u003c/sup\u003e exo, R47H\u003csup\u003ehom\u003c/sup\u003e exo, TREM2\u003csup\u003e-/-\u003c/sup\u003e exo, respectively). The exosomes presented with the expected size at the range of 200 nm and expressed the exosomal markers tested (\u003cstrong\u003eFigure 1c\u003c/strong\u003e) (as we also determined previously; Mallach et al., 2021). To explore the effect of Cv, R47H\u003csup\u003ehet\u003c/sup\u003e, R47H\u003csup\u003ehom\u003c/sup\u003e TREM2\u003csup\u003e-/-\u003c/sup\u003e iPSC-microglia secreted exosomes on neuronal gene expression profiles, we performed RNA-seq in iPSC-neurons incubated with microglia exosomes for 48 h. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Neuronal gene expression changes due to exosomal treatment from different TREM2 microglial lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential expression analyses showed altered expression of a number of genes including \u003cem\u003eSULF1\u003c/em\u003e, \u003cem\u003eMMP2\u003c/em\u003e, \u003cem\u003ePLD1\u003c/em\u003e between untreated neurons and neurons treated with TREM2 Cv exosomes (\u003cstrong\u003eFigure 2a\u003c/strong\u003e). These results were validated by qPCR where a panel of 6 genes were selected that showed significance in one of the comparisons shown throughout Figures 2 and 3: \u003cem\u003eSULF1\u003c/em\u003e, \u003cem\u003eMMP2\u003c/em\u003e, \u003cem\u003eSSPO, IL33\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;NPTX2\u003c/em\u003e (\u003cstrong\u003eFigure 2b, Supplementary figure S1\u003c/strong\u003e). The genes differentially expressed due to Cv microglial exosomes were significantly enriched for biological annotations associated with extracellular matrix and structure organization, cell migration and, glia cell derived neurotrophic factor (GDNF) receptor signalling pathway and\u0026nbsp;Wingless-related integration site (Wnt) signalling pathway (\u003cstrong\u003eFigure 2c\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA higher number of genes showed increased expression in neurons treated with R47H\u003csup\u003ehet\u003c/sup\u003e patient-derived microglial exosomes including 69 genes with altered expression in total (37 increased, 32 decreased) (\u003cstrong\u003eFigure 2d, Supplementary figure S1\u003c/strong\u003e). Among them, the genes that were found increased were associated with metabolic pathways including cellular and mitochondrial fatty acid metabolism, and activity of related enzymes and channel activities. The genes with decreased expression were associated with growth factor complex (\u003cstrong\u003eFigure 2f\u003c/strong\u003e). Interestingly, \u003cem\u003eSULF1\u003c/em\u003e, \u003cem\u003eMMP2\u003c/em\u003e, \u003cem\u003eIL33\u003c/em\u003e and \u003cem\u003eNPTX2\u003c/em\u003e genes were found to be significantly increased by qPCR compared with control untreated neurons \u003cstrong\u003e(Figure 2e).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe found 53 genes differentially expressed in the neurons due to R47H\u003csup\u003ehom\u003c/sup\u003e-derived microglial exosomes with 29 upregulated and 24 downregulated (\u003cstrong\u003eFigure 2g, Supplementary figure S1\u003c/strong\u003e). The upregulated genes were involved in synapse organization and density as well as pyruvate metabolic processes whereas the\u0026nbsp;downregulated genes were associated with lipid metabolic processes and extracellular matrix organization (\u003cstrong\u003eFigure 2i\u003c/strong\u003e).\u0026nbsp;Likewise, we found 24 genes differentially expressed in neurons following incubation with TREM2\u003csup\u003e-/-\u003c/sup\u003e-derived microglial exosomes with 7 genes upregulated and 17 genes downregulated \u003cstrong\u003e(Figure 2j, Supplementary figure S1).\u0026nbsp;\u003c/strong\u003eThese genes were associated with lactate and pyruvate metabolic processes and matrix extracellular assembly (upregulated) and cerebellar atrophy, aggressive behavior and epileptic conditions (downregulated) \u003cstrong\u003e(Figure 2l).\u003c/strong\u003e \u003cem\u003eSULF1\u003c/em\u003e, \u003cem\u003eMMP2, IL33, NPTX2,\u0026nbsp;\u003c/em\u003egenes in TREM2\u003csup\u003e-/-\u003c/sup\u003e exosome treated neurons were not significantly altered compared to control untreated neurons \u003cstrong\u003e(Figure 2k).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Exosome induced neuronal gene changes due to microglial R47H expression or TREM2 loss.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore alterations in a landscape more relevant to brain physiology, we performed differential expression analysis between Cv microglia exosome-treated neurons and R47H\u003csup\u003ehet\u003c/sup\u003e patient (118 total genes: 67 upregulated, and 51 downregulated) or TREM2\u003csup\u003e-/-\u003c/sup\u003e (90 total genes: 30 upregulated and 60 downregulated) microglia secreted exosomes. This analysis revealed a 2 to 5-fold increase in the number of differentially expressed genes in the selected conditions. More specifically 118 neuronal genes were shown to be differentially expressed due to exosomes derived from R47H\u003csup\u003ehet\u003c/sup\u003e iPSC-microglia (\u003cstrong\u003eFigure 3a and b\u003c/strong\u003e). The genes upregulated in neurons were involved in neuronal ensheathment, myelin assembly and intracellular signalling pathways, whereas the downregulated neuronal genes were associated with nitrite-related processes, and cellular amino-acid metabolic processes (\u003cstrong\u003eFigure 3c\u003c/strong\u003e). Similarly, 90 neuronal genes were shown to be differentially expressed in neurons treated with exosomes secreted by microglia lacking TREM2 vs neurons treated with Cv microglial exosomes (\u003cstrong\u003eFigure 3d and e\u003c/strong\u003e). The genes upregulated in TREM2\u003csup\u003e-/-\u003c/sup\u003e exosome-treated neurons were involved in mitochondrial respiration and cellular and oxidative phosphorylation, and pathways associated with neurodegeneration and AD, whereas the downregulated genes were involved in annotations associated with neurogenesis and neuronal differentiation and brain development (\u003cstrong\u003eFigure 3f\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.\u003c/strong\u003e \u003cstrong\u003eExosomal effects on neuronal metabolism\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the observed impact of exosomes on the neuronal transcriptome related to metabolic processes, we explored the direct effect of the exosomes from TREM2 variants on neuronal oxidative phosphorylation (OXPHOS) by Seahorse analysis. Microglial exosomes did not significantly affect the spare respiratory capacity or ATP productions of the cells (\u003cstrong\u003eFigure 3g-i\u003c/strong\u003e); however, we observed a significant decrease in coupling efficiency and increased proton leak accordingly, in neurons incubated with exosomes secreted by microglia lacking TREM2 when compared with neurons treated with exosomes from Cv microglia (\u003cstrong\u003eFigure 3j-k\u003c/strong\u003e), confirming the transcriptomic results. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Exosomal effects on neurite outgrowth at basal and A\u0026beta;\u003csub\u003e1-42\u0026nbsp;\u003c/sub\u003einduced damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effect of exosomes on the neurite network in our iPSC model we analysed the neuronal outgrowth. At basal conditions, iPSC-neurons treated with R47H\u003csup\u003ehet\u003c/sup\u003e exosomes exhibited decreased neuronal outgrowth which did not reach significance when data from patient 1 (D, dementia) and 2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(nonD; non-dementia) were pooled. However, when we analysed separately data from patient 1 and 2, statistical analysis revealed a significant reduction of neurite outgrowth (max and total neurite length) and number of roots in neurons treated with exosomes from patient 1 (dementia patient) compared with untreated neurons (\u003cstrong\u003eFigure 4a and 4c-e\u003c/strong\u003e) at basal conditions, and did not protect against A\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e -toxicity (Figure 4c and d). Likewise, neurons treated with TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes presented a decreased neurite length at basal conditions and upon A\u0026beta;\u003csub\u003e1-42\u0026nbsp;\u003c/sub\u003etreatment TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes were not protective (\u003cstrong\u003eFigure 4d and e\u003c/strong\u003e). A\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e treatment decreased neurite length, and interestingly, the addition of Cv exosomes, but not R47H\u003csup\u003ehet\u003c/sup\u003e or TREM2\u003csup\u003e-/-\u003c/sup\u003e derived exosomes showed a trend toward prevention, with a borderline statistical significance (\u003cstrong\u003eFigure 4a, c\u003c/strong\u003e). Similarly, the number of neurite roots was decreased in neurons treated with TREM2 variant derived exosomes compared to neurons treated with Cv exosomes upon A\u0026beta;\u0026nbsp;stimulation (\u003cstrong\u003eFigure 4c\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Exosomal effects on synaptic function and A\u003c/strong\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003e1-42\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;related synaptic dysfunction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess whether microglia exosomes deliver neuroprotective or detrimental effects at a synaptic level, we evaluated the expression of the presynaptic (SYN) and postsynaptic (PSD95) markers in our iPSC-neuron model. As expected\u0026nbsp;\u0026Alpha;\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e oligomers decreased significantly the expression of SYN (\u003cstrong\u003eFigure 4f\u003c/strong\u003e). Cv exosomes were shown to protect \u0026Alpha;\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e challenged neurons in terms of their SYN expression, but R47H\u003csup\u003ehom\u003c/sup\u003e or TREM2\u003csup\u003e-/-\u003c/sup\u003e (\u003cstrong\u003eFigure 4f\u003c/strong\u003e) or R47H\u003csup\u003ehet\u0026nbsp;\u003c/sup\u003e(\u003cstrong\u003eFigure 4g\u003c/strong\u003e) derived exosomes did not. Notably, R47H\u003csup\u003ehet\u003c/sup\u003e from dementia patient 1 and TREM2\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003emicroglial-secreted exosomes were shown to decrease basal levels of SYN expression (\u003cstrong\u003eFigure 4f-g\u003c/strong\u003e) which were not further decreased by\u0026nbsp;\u0026Alpha;\u0026beta;\u003csub\u003e1-42\u0026nbsp;\u003c/sub\u003etreatment. Microglial exosomes did not significantly alter PSD95 expression at basal (\u003cstrong\u003eFigure 4h\u003c/strong\u003e) or A\u0026beta; treated neurons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test whether exosomal incubation affected glutamate release from neurons and whether TREM2 variant-derived exosomes altered this, we evaluated the levels of glutamate in neuronal supernatant after incubation with exosomes. Cv exosomes led to a significant increase in glutamate levels, and this increase was not detected following incubations with exosomes derived by TREM2 variant or TREM2\u003csup\u003e-/-\u003c/sup\u003e microglia (\u003cstrong\u003eFigure 4i\u003c/strong\u003e), suggesting that these exosomes are not able to promote glutamate release and therefore a proper neuronal function.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7. Effect of exosomes on cell stress proteome and neuronal apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we assessed how TREM2 variant exosomes might affect the neuronal proteome with regard to cell stress (\u003cstrong\u003eFigure 5a-c\u003c/strong\u003e). Exosomes from Cv microglia led to an overall slight decrease in the expression of cell stress-related proteins in the neurons, such as Carbonic Anydrase IX, Cytochrome C, Hypoxia-Inducible factor -1\u0026alpha; (HIF-1\u0026alpha;), and Nuclear factor kappa B (NF-kB) (\u003cstrong\u003eFigure 5b and c\u003c/strong\u003e) and in turn, the expression of those showed a trend toward an increase upon incubation with R47H or TREM2\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003emicroglia derived exosomes (\u003cstrong\u003eFigure 5b and c\u003c/strong\u003e).\u0026nbsp;By evaluating the expression levels of cleaved caspase-3 we were able to determine whether TREM2 variant microglia exosomes promoted neuronal apoptosis. Our results showed an increasing trend in expression in neurons treated with TREM2 variant microglia exosomes, although it did not reach significance (\u003cstrong\u003eFigure 5e\u003c/strong\u003e). Upon an \u0026Alpha;\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e challenge of 5 \u0026mu;M we did not observe any further induction of apoptosis, but consistently, an increasing trend of cleaved caspase-3 expression in neuronal groups treated with TREM2 variant exosomes was revealed (\u003cstrong\u003eFigure 5f\u003c/strong\u003e).\u0026nbsp;To test whether exosomes affect neuronal viability at the concentration added, and thus, whether the observed changes at transcriptomic or functional (metabolic, synaptic or apoptotic) level were due to cell death we assessed the neuronal viability which was not altered within the 48 treatment window (\u003cstrong\u003eFigure 5g\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur data show that exposure of iPSC-derived cortical neurons to exosomes isolated from iPSC-microglia expressing common variant TREM2, the LOAD risk variant R47H\u003csup\u003ehet\u003c/sup\u003e or the R47H\u003csup\u003ehom\u003c/sup\u003e and TREM\u003csup\u003e-/-\u003c/sup\u003e lines have significant and differing effects on the neuronal genome. In particular the genes affected are altered in different ways by the different line-derived exosomes. These findings extend our previous work in which we found that the exosomes themselves from different TREM2-variant iPSC-microglia show differential protein expression profiles and influenced SHSY5Y survival (Mallach et al., 2021a; b), again in a manner in which the TREM2 AD variant line-derived exosomes were not supportive of survival when compared with the Cv line-derived exosomes.\u003c/p\u003e\n\u003cp\u003eExosomal signaling (and that of larger extracellular vesicles, EVs) is becoming an increasingly important pathway in intercellular signaling (Hill, 2019; Wang and Xia, 2022). Exosomes are the most widely studied EVs and are derived from endosomes. We showed previously that microglial-derived exosomes can be taken up by neurons (Mallach et al., 2021a), suggesting this may be the route by which they can influence neuronal behaviour (Hooper et al., 2012). Interestingly, as with our findings for microglial-derived exosomes, oligodendrocytes can also release exosomes, which have been found to contain a number of proteins associated with protection against cell stress, and which has been argued could provide axonal support against injury (Kr\u0026auml;mer‐Albers et al., 2007). However to date, many of these studies have been performed on rodent derived exosomes\u0026nbsp;(Hooper et al., 2012; Xie et al., 2022). These authors showed that astrocytes influenced\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eneurodegenerative diseases through metabolic balance and ubiquitin-dependent protein balance, whereas microglial-derived exosomes influenced neurodegenerative diseases through immune inflammation and oxidative stress. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicroglia exosomes have been shown to mediate neuroprotective or neurotoxic effects in a context dependent manner. In this study, healthy microglia exosomes were shown to primarily alter neuronal genes involved in extracellular matrix and structure organization, cell migration and motility. Extracellular matrix components which were upregulated by exosomal treatments such as \u003cem\u003eSULF1\u003c/em\u003e, \u003cem\u003eMMP2\u003c/em\u003e or \u003cem\u003eCOL1A1\u003c/em\u003e have been reported to play a role in the formation, maintenance and function of synapses (Kalus et al., 2015; Condomitti and De Wit, 2018; Kamimura and Maeda, 2021) or associated with A\u0026beta; pathology and \u0026nbsp;AD (Hosono-Fukao et al., 2012; Hernandez-Guillamon et al., 2015; Ozsan McMillan et al., 2023), further supporting a role of exosomes in neuronal support. TREM2 variant microglia derived exosomes also affected the expression of selected related genes when added to neurons, suggesting that this effect may be primarily mediated by changes in exosomal content due to TREM2 variants, and not directly related/altered due to TREM2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn line with the previously observed alterations in exosomal cargo related to metabolic processes due to R47H\u003csup\u003ehet\u003c/sup\u003e (Mallach et al., 2021b), here, R47H\u003csup\u003ehet\u003c/sup\u003e, R47H\u003csup\u003ehom\u003c/sup\u003e and TREM2\u003csup\u003e-/-\u003c/sup\u003e microglia derived exosomes were shown to affect the human neuronal transcriptome associated with metabolic processes including lipid or lactate metabolism. Comparative analysis between R47H or TREM2\u003csup\u003e-/-\u003c/sup\u003e and Cv exosome treated neurons further revealed differences in the expression of genes involved in various metabolic processes, including amino-acid metabolic pathways, oxidative phosphorylation and mitochondrial complexes confirming that the observed alterations can be attributed to TREM2 altered function or absence. Recently, it was shown that TREM2\u003csup\u003e-/-\u003c/sup\u003e impairs neuronal transcriptome and bioenergetics \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003e(Tagliatti et al., 2024) and conditioned media from microglia expressing R47H decreased OXPHOS in human neurons (Vasilopoulou et al., 2024a). Thus, the present data suggest that these effects are mediated via mechanisms linked to exosomal pathways that are influenced by TREM2. Indeed, when TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes were directly added to neurons, coupling efficiency and ATP production in neurons declined indicating mitochondrial dysfunction. In our experimental setting R47H exosomes did not significantly affect OCR parameters in real time metabolic analysis; however the observed trends follow the trend of TREM2 loss level (het\u0026gt;hom\u0026gt;absent). Interestingly, a set of genes altered via exosomes derived from TREM2 deficient microglia (compared with Cv exosomes) was associated with TCA cycle and respiratory electron transport. We previously showed that fueling microglia with citrate or succinate, two key TCA components, can rescue certain functional deficits due to R47H or TREM2\u003csup\u003e-/-\u003c/sup\u003e and improve neuronal metabolic function via the secretome (Vasilopoulou et al., 2024a). This evidence suggests that exosomes, acting as key microglial secretome components, may be responsible for the observed effects, and additionally, ameliorating TREM2 deficient signalling in microglia and subsequently impaired metabolism may prevent negative exosome-mediated effects, due to TREM2 deficiency, on neuronal metabolism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother set of differentially expressed genes pointed to alterations at synaptic processes and neuronal transmission delivered by microglial exosomes and influenced by TREM2 state, including axon and neuronal ensheathment, myelination, postsynaptic density and synapse organisation or neurogenesis. Previous research has shown an important role of microglia-derived EVs on synaptic plasticity and dysfunction (Gabrielli et al., 2022). These effects have been attributed in some cases on exosomal cargo of packaged miRNAs e.g. miR-223 enriched exosomes reduced neuroinflammation and ameliorated nerve damage in AD \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e (Wei et al., 2024). Among the differentially expressed genes neuronal \u003cem\u003eNPTX2\u003c/em\u003e was dysregulated in response to R47H\u003csup\u003e\u0026nbsp;\u003c/sup\u003ederived exosomes. \u003cem\u003eNPTX2\u003c/em\u003e regulates complement activity and microglial synapse elimination in the brain (Zhou et al., 2023) and is considered a protective mechanism upon neuronal damage. However, recent findings have implicated NPTX2 in TDP-43-induced neurodegeneration, which characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in a human model of long-lived mature neurons\u0026nbsp;(iPSC- colony morphology neural stem cells),\u0026nbsp;suggesting a neurotoxic role under certain conditions\u0026nbsp;(Hruska-Plochan et al., 2024). In this context, the neuronal dysregulation of \u003cem\u003eNPTX2\u003c/em\u003e may reflect a neuronal response following synaptic damage induced by exosomes.\u0026nbsp;Indeed, post-translationally, exosomes derived from unstimulated variant microglia or microglia lacking TREM2 not only were less efficient at providing neuronal support but instead neurite outgrowth was decreased by LoF variant derived exosomes. Whether \u003cem\u003eNPTX2\u0026nbsp;\u003c/em\u003edysregulation in this setting contributes to a protective or a pathogenic response, as observed in ALS and FTD models deserves further exploration. Moreover, microglial exosomes promoted glutamate release which reflects a physiological neuronal function as previously shown\u0026nbsp;(Antonucci et al., 2012)\u0026nbsp;whereas exosomes lacking TREM2 did not. Consistently, A\u0026beta;-induced damage of the neural network was mitigated by Cv microglia exosomes but R47H or TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes were unable to deliver any protective effects at such conditions. These data suggest a crucial role of TREM2 on neuronal health.\u003c/p\u003e\n\u003cp\u003eAccordingly, at a synaptic level, Cv exosomes protected A\u0026beta;-induced synaptic damage but when TREM2 was deficient they did not. Interestingly, the synaptic effects of R47H exosomes were stronger when derived from R47H\u003csup\u003ehet\u003c/sup\u003e carrier 1 who exhibited dementia in contrast to R47H\u003csup\u003ehet\u003c/sup\u003e carrier 2 that did not manifest dementia symptoms, suggesting a potential link between the observed exosomal effect \u003cem\u003ein vitro\u003c/em\u003e and cognitive state at a clinical level (Duggan et al., 2022) or reflecting background genomic variability between carriers. This observation aligns with GO enrichment annotations implicating TREM2 loss with diseases such as AD and PD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhether the observed effects are due to the absence of \u0026ldquo;protective\u0026rdquo; cargo when TREM2 is deficient/absent or the presence of \u0026ldquo;toxic\u0026rdquo; cargo that is increased in exosomes released by TREM2\u003csup\u003e-/-\u003c/sup\u003e microglia or both needs further exploration. It has been shown that under pathophysiological conditions exosomes can transfer pro-inflammatory damage associated molecular patterns (DAMPs) to surrounding cells. To further assess whether TREM2 LoF or absence compromises any beneficial/protective effects of microglial exosomes on neurons \u0026ndash; or instead promotes detrimental effects, we investigated the cell stress-related neuronal proteome following microglial exosomal exposure. Overall, TREM2 loss was shown to prime cell stress related proteins (e.g HIF-1\u0026alpha;, HIF-2\u0026alpha;, Paraoxonases (PON) involved in metabolic/oxidative stress processes, which have been shown to be regulated by TREM2 in microglia cells (Ulland et al., 2017; March-Diaz et al., 2021). TREM2 R47H variant or TREM2\u003csup\u003e-/-\u003c/sup\u003e exosomes seem to promote subtle apoptotic effects on neurons and further contribute to neuronal apoptosis when neurons are primed with A\u0026beta;\u003csub\u003e1-42\u003c/sub\u003e; however, exosomal treatments do not affect cell viability by apoptosis over the time course of our experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe previously found that the R47H\u003csup\u003ehet\u003c/sup\u003e-microglia display mitochondrial bioenergetic changes, with an inability to switch to glycolysis (Piers et al., 2020) and an inability to activate the inflammasome (Cosker et al., 2021). We show in the current work that neuronal oxidative phosphorylation is also altered by TREM2 LoF variants, which would likely impact on neuronal signalling responses. With regard to the inflammasome, a recent report suggests the inflammasome may facilitate exosome secretion of neurotoxic species such as\u0026nbsp;a-synuclein in PD\u0026nbsp;(Si et al., 2021). However whilst we cannot definitively say that a reduced inflammasome activity per se in R47H\u003csup\u003ehet\u0026nbsp;\u003c/sup\u003eiPSC-microglia directly influences exosome content (because we did not stimulate Cv or R47H microglia in a manner which would activate the inflammasome)\u0026nbsp;(Cosker et al., 2021; Mallach et al., 2021b)\u0026nbsp;we have shown that TREM2 LoF do produce an exosome population which is significantly different from Cv.\u0026nbsp;It is also worth noting that the effects of the exosomes on neurons were assessed over a relatively short period of time. Even within that timeframe, we were able to detect a measurable degree of change. Future studies should aim to investigate the longer-term consequences of microglial exosomal exposure on neuronal function and viability.\u003c/p\u003e\n\u003cp\u003eCellular functional consequences due to TREM2 impairment in microglia extend to neurons, but the precise intercellular mechanisms are not determined. Here, we show that TREM2 can shape the effects of human microglial exosomes on neuronal transcriptome profile and functions. Our data provide insights into the intercellular exosomal pathways whereby microglia affect neuronal heath in a TREM2-dependent manner. Taken together, our data suggest that alongside the direct cell-to-cell contact and the soluble factor release, microglia can communicate with neurons by bidirectional release of exosomes. Microglial exosomes contain TREM2 \u0026nbsp;as part of their cargo (Mallach et al., 2021b; Huang et al., 2022) suggesting a role of TREM2 on subsequent exosomal mediated effects. There is likely to be further bi-directional cross talk and increased complexity following the involvement of oligodendrocytes and astrocytes in these studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD, Alzheimer\u0026rsquo;s disease; ALS, amyotrophic lateral sclerosis; bFGF, basic fibroblast growth factor; EVs, Extracellular vesicles; FTD, frontotemporal dementia; GDNF, glia cell derived neurotrophic factor; iPSC, induced pluripotent stem cell; LOAD, Late-onset Alzheimer\u0026rsquo;s disease; LoF, loss-of-function; NIM, neuronal induction media; NMM, neuronal maintenance media; NPC, neuronal progenitor cells; PLO, poly-L-ornithine; TREM2, triggering receptor expressed on myeloid cells-2; Wnt, Wingless-related integration site \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding statement:\u0026nbsp;\u003c/strong\u003eWe acknowledge the Rosetrees Trust, UK for providing funding for the salary of FV and research costs of this project, (Funding to JMP, SW and DS). We acknowledge support for DS from the Dolby Foundation, USA (to JH) and the Dementia Research Institute, University College London (to JH). SW and JH are supported by the UCLH NIHR Biomedical Research Centre.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eJ.M.P. and F.V. designed the study, F.V. carried out the experiments and analysed the data, U.Y. and D.A.S. performed the RNA-seq bioinformatics analysis, J.M.P and F.V. wrote the paper. \u0026nbsp;All authors provided comments, contributed to and agreed on the final version of the paper. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u0026nbsp;\u003c/strong\u003eWe thank Andrew Weston, UCL School of Pharmacy for help with the electron microscopy images of exosomes.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u0026nbsp;\u003c/strong\u003eAll data are available upon request. The RNA-seq data have been deposited in the Gene Expression Omnibus (GEO).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAntonucci F, Turola E, Riganti L, Caleo M, Gabrielli M, Perrotta C, Novellino L, Clementi E, Giussani P, Viani P, Matteoli M, Verderio C. 2012. Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism: Microglial MVs increase sphingolipid metabolism in neurons. The EMBO Journal 31:1231\u0026ndash;1240.\u003c/li\u003e\n \u003cli\u003eCondomitti G, De Wit J. 2018. Heparan Sulfate Proteoglycans as Emerging Players in Synaptic Specificity. Front Mol Neurosci 11:14.\u003c/li\u003e\n \u003cli\u003eCosker K, Mallach A, Limaye J, Piers TM, Staddon J, Neame SJ, Hardy J, Pocock JM. 2021. Microglial signalling pathway deficits associated with the patient derived R47H TREM2 variants linked to AD indicate inability to activate inflammasome. 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Mol Neurodegeneration 17:58.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"exosomes, microglia, neurons, TREM2 R47H AD risk variant, neurodegeneration","lastPublishedDoi":"10.21203/rs.3.rs-7487794/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7487794/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroglial exosomes are key secretome components that modulate cell-to-cell communication mediating protective or detrimental effects depending on the environmental context upon release. The R47H variant in the microglial triggering receptor expressed on myeloid cells-2 (TREM2) increases the risk for late-onset Alzheimer\u0026rsquo;s disease (AD) and influences microglia function, contributing to neurodegeneration. Our group has previously shown that the proteome content of exosomes released by microglia harboring the TREM2 R47H mutation differs from that of TREM2 common variant microglial exosomes, suggesting an altered microglia-neuron interaction and neuronal function upon their secretion. To further investigate how R47H variants or TREM2 loss modifies exosome effects on neurons, we assessed further their effects on human iPSC-neurons. We assessed transcriptome and proteome changes in neurons using RNA-seq and proteomic analyses. Our findings reveal that exosomes secreted by R47H variant iPSC-microglia differentially regulate the neuronal transcriptome associated with metabolic pathways and synaptic function and the observed changes in the cell stress-related proteome of neurons further supports this. Additionally, we provide evidence regarding the effects of these exosomes on pre-synaptic and post-synaptic and apoptotic markers expressed by our neuronal model and the influence of TREM2 status on synaptic functioning. Collectively, our data contribute to the characterization of human microglial exosome functions and provide novel insights into how this emerging communication pathway is affected by TREM2 late-onset AD risk variants.\u003c/p\u003e","manuscriptTitle":"Impact of iPSC-derived microglial exosomes on neurons: Role of TREM2 and implication in Alzheimer's Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 13:14:16","doi":"10.21203/rs.3.rs-7487794/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd3dcd9d-bada-4df2-930d-d497e147b8d8","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-02T01:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 13:14:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7487794","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7487794","identity":"rs-7487794","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}