Alzheimer's disease (AD) remains the leading cause of dementia worldwide and currently lacks effective therapies. Recent studies have identified AD-associated genetic variants in several microglial-enriched genes, highlighting microglia as key contributors to disease pathogenesis and as promising therapeutic targets. These include several risk variants in Trem2 and a rare coding risk variant in Abi3 (rs616338:p. Ser209Phe). The impact of ABI3 deletion on AD pathology remains inconclusive as recent studies in ABI3-deficient AD mouse models have produced conflicting observations. Specifically, the deletion of ABI3 reduces amyloid phenotypes in the TgCRND8 transgenic mouse model but exacerbates these phenotypes in the 5XFAD mouse model. We hypothesize that TREM2 signalling differences in C57 (genetic background of TgCRND8) and SJL (genetic background of 5XFAD) microglia contribute to this discrepancy. Using primary mouse microglia derived from C57 and SJL mice, we analyzed TREM2 cleavage by ELISA and TREM2 signalling by stimulating the cells using an activating antibody and measuring SYK and PLCG2 phosphorylations (validated components of TREM2 signalling pathway) by western blotting. In addition, we compared TREM2-dependent phagocytosis of amyloid-beta by C57 and SJL microglia using IncuCyte S3 live imaging. We observed significant alterations in TREM2 signalling in SJL microglia compared to C57 microglia. TREM2 cleavage was significantly lower in SJL microglia and following stimulation by an anti-TREM2 activating antibody, SJL microglia demonstrated attenuated SYK and PLCG2 phosphorylations and reduced amyloid-beta phagocytosis compared to C57 microglia. We have demonstrated that TREM2 signalling and the related AD-associated functions are significantly altered in SJL microglia. In addition, previous co-expression network analyses have suggested a close functional relationship between TREM2 and ABI3. A combined presence of altered TREM2 signalling and ABI3 deletion may explain the diverging phenotypes observed in TgCRND8 and 5XFAD mouse models. We are currently investigating other potential contributing factors such as differences in genetic background and AD transgenes expressed in these mouse models. We aim to uncover the molecular machinery that links TREM2 to ABI3 in microglia and to understand the impact of ABI3 S209F mutation on TREM2 signalling in microglia and in AD pathology, pathing the way towards identifying novel therapeutics.

Disease-associated microglia (DAM), initially described in mouse models of neurodegenerative diseases, have been classified into two related states; starting from a TREM2-independent DAM1 state to a TREM2dependent state termed DAM2, with each state being characterized by the expression of specific marker genes (Keren-Shaul, 2017). Recently, single-cell (sc)RNA-Seq studies have reported the existence of DAM in humans (Pettas, 2022; Jauregui, 2023; Friedman, 2018; Mathys, 2019; Tuddenham, 2024); however, whether DAM play beneficial or detrimental roles in the context of neurodegeneration is still under debate (Butovsky and Weiner, 2018; Wang and Colonna, 2019). Here, we present a pharmacological approach to mimic human DAM in vitro: we validated in silico predictions that two different histone deacetylase (HDAC) inhibitors, Entinostat and Vorinostat, recapitulate aspects of the DAM signature in two human microglia-like model systems. HDAC inhibition increases RNA expression of MITF, a transcription factor previously described as a regulator of the DAM signature (Dolan, 2023). This engagement of MITF appears to be associated with one part of the DAM signature, refining our understanding of the DAM signature as a combination of at least two transcriptional programs that appear to be correlated in vivo. Further, we functionally characterized our DAM-like model system, showing that the upregulation of this transcriptional program by HDAC inhibitors leads to an upregulation of amyloid β and pHrodo Dextran uptake - while E.coli uptake is reduced - and a specific reduction of MCP1 secretion in response to IFN-γ and TNF-α. Enhanced amyloid β uptake was confirmed in iPSC-derived microglia. Overall, our strategy for compound-driven microglial polarization offers potential for exploring the function of human DAM and for an immunomodulatory strategy around HDAC inhibition.

The sialic-acid binding immunoglobulin-like lectin 3 receptor (Siglec-3 / CD33) is one of the highly associated AD risk genes. Previous studies revealed that the non-coding AD-risk alleles (rs3865444 and rs12459419) are associated with increased total levels of CD33 expression and a higher relative expression of the long CD33 M splice form. However, the molecular basis of the immuno-inhibitory function of CD33 remains unclear. To confirm the presence of CD33 dimers, we conducted multiple experiments. Blue Native Gel electrophoresis and co-immunoprecipitation assays were used to detect CD33 bands corresponding to the expected molecular weights. Flow cytometry with specific antibodies was performed to quantify cell-surface CD33. Additionally, single-molecule fluorescence resonance energy transfer (smFRET) combined with TIRF imaging was employed to visualize CD33 dimers on the cell surface. Furthermore, Western Blotting of phosphorylation of CD33 and its downstream molecule were performed to verify if the dimers were functional. Biochemical analyses demonstrated that CD33 M and CD33 m can form homodimers or heterodimers. Flow cytometry confirmed that CD33 M isoforms are selectively trafficked to the cell surface, while smFRET imaging verified the presence of dimers on the cell surface. The elevation of the CD33 pathway following stimulation with CD33-specific ligands provided evidence that CD33 M homodimers are functional. This study reveals the critical role of CD33 M in AD pathology by elucidating its molecular mechanisms. We provide direct evidence that CD33 M and CD33 m isoforms can form both homodimers and heterodimers. However, only CD33 M isoforms are preferentially trafficked to the cell surface and form functional dimers. These findings advance our understanding of the molecular basis of CD33's immuno-inhibitory function and offer new insights into its involvement in AD risk, potentially paving the way for the development of targeted therapeutic strategies.

Missense mutations in the TREM2 gene are associated with increased risk of Alzheimer's disease (AD). Aβ is a known ligand of TREM2 binding directly and activating a signalling pathway involving PLCG2. TREM2 itself signals through its association with DAP12 and recruits SYK through its cytosolic immune-receptor tyrosine-based activation motifs. This study will provide insights into relevant TREM2-PLCG2-associated cellular processes to help to identify novel interacting proteins and dissect cellular mechanisms involved in AD. Aβ and anti-TREM2 activating antibody were used to stimulate TREM2 signalling in mouse microglia followed by western blotting to interrogate the phosphorylation state of various interactors under basal and stimulated conditions. Co-IP and Immunofluorescence studies were performed to investigate protein interactors in this signalling cascade. Migration and Aβ engulfment assays were used as downstream functional readouts of TREM2 stimulation. Results are representative of at least three independent biological replicate experiments. We discovered that the endogenous TREM2-DAP12-PLCG2 signaling complex interacts with numerous novel high affinity components in microglia. We found that the levels of phospho-SYK (Tyr525/526) and non-canonical phospho-tyrosines in PLCG2 were significantly increased by TREM-2 activation. Increased Phospho-PLCG2 levels triggered changes in microglia function reflected in functional assays such as cell migration and cargo engulfment assays. We discovered that stimulation of TREM2 in microglia led to a novel signalling cascade involving PLCG2 and other proteins which mediated important microglia functions. Some of these proteins are components of the B cell receptor signaling machinery acting via PLCG2 while others are known/new AD-related risk genes. The elucidation of the TREM2-PLCG2 pathway and its downstream signalling partners provides important insights into the mechanisms of normal microglia function and in the pathogenesis of AD.

We report the results of structural, functional and genetic studies on the CD33 sialic acid- binding receptor that reveal how non-coding variants in CD33 alter risk for Alzheimer's disease (AD). The full-length CD33 M isoform, whose expression is upregulated by non-coding AD-risk alleles, preferentially forms dimers at the cell surface, where they interact with AD-related proteins (clusterin and Aβ). This interaction induces CD33 M inhibitory signalling and downregulates protective microglial functions including phagocytic removal of amyloid plaques. Human brain expression quantitative trait loci (eQTL) and causal mediation analyses confirm that quantitative interactions between CLU and CD33 genotypes modulate AD phenotypes and suggest that genotypes at these loci might be used to personalise future therapeutic approaches. Our work also highlights several other unexpected aspects of CD33 biology, including a soluble shed extracellular fragment of CD33 M and a similar soluble secreted product arising from a truncating mutation in the CD33 extracellular domain (CD33 MΔ4bp ).

The sialic-acid binding immunoglobulin-like lectin 3 receptor (Siglec-3 / CD33) expressed on microglia, regulates immune functions relevant to Alzheimer's disease (AD). Clusterin (CLU) and apolipoprotein E (ApoE) are soluble, sialylated proteins implicated in AD pathogenesis through genetic associations and their interactions with amyloid-beta (Aβ). However, the role of these proteins as potential CD33 ligands remains unclear. This study explores whether CLU and/or ApoE bind CD33 and examines the functional impact of these interactions on Aβ uptake and amyloid plaque clearance. The binding of CD33 to CLU and ApoE was assessed through co-immunoprecipitation using U937 cells (endogenously expressing CD33) and HEK293 cells (expressing exogenous CD33). Quantitative bio-layer interferometry (BLI) and microscale thermophoresis determined binding affinities, focusing on the role of CD33's Arg119 sialic acid binding site. In situ proximity ligation assays (PLA) and co-immunoprecipitation from AD and control human brain lysates validated in vivo interactions. Functional assays examined Aβ uptake and amyloid plaque clearance in monocytes and U937 cells, with or without CLU treatment. The quantitative binding assay revealed that CLU, but not ApoE, was a sialylation-dependent ligand for CD33, binding with high affinity (Kd = 28.9 ± 10.3 nM). Binding required an intact Arg119 residue and dimeric CD33 structure. PLA and co-immunoprecipitation studies demonstrated colocalization of CD33 and CLU on microglia in AD brains, especially near amyloid plaques. Functionally, sialylated CLU inhibited Aβ uptake in monocytes from CD33 “CC” risk allele carriers and reduced amyloid plaque clearance in U937 cells. Desialylated CLU showed no significant effects. Notably, CLU + Aβ oligomers induced stronger CD33 ITIM signaling than CLU alone, enhancing phosphorylation and SHP-1 recruitment. This study identifies CLU as a specific CD33 ligand and highlights its role in modulating microglial functions via CD33 ITIM signaling. Sialylated CLU inhibits Aβ uptake and amyloid plaque clearance, suggesting a potential mechanism underlying microglial dysfunction in AD. These findings underscore the therapeutic potential of targeting the CD33-CLU axis to restore microglial homeostasis and enhance amyloid clearance in AD.

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aβ and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aβ oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aβ oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aβ oligomerization and disaggregated preformed Aβ oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aβ fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aβ-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aβ but rather promotes Aβ protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aβ-induced release of sTREM2, which blocks Aβ aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aβ aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.

Missense mutations (e.g. R47H) of the microglial receptor TREM2 increase risk of Alzheimer’s disease (AD), and the soluble ectodomain of wild-type TREM2 (sTREM2) appears to protect in vivo, but the underlying mechanisms are unclear. We show that Aβ oligomers bind to TREM2, inducing shedding of sTREM2. Wild-type sTREM2 inhibits Aβ oligomerization, fibrillization and neurotoxicity, and disaggregates preformed Aβ oligomers and protofibrils. In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aβ, but rather promotes Aβ protofibril formation and neurotoxicity. Thus, in addition to mediating phagocytosis, wild-type TREM2 may protect against amyloid pathology by Aβ-induced release of sTREM2 that blocks Aβ aggregation and neurotoxicity; while R47H sTREM2 promotes Aβ aggregation into neurotoxic forms, which may explain why the R47H variant gene increases AD risk several fold.

We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild-type and H157Y human TREM2 and for the wild-type murine orthologue. Crucially, we also show that the Alzheimer’s disease-associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastatand ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer’s and other neurological diseases.

Traditional drug development for Alzheimer's disease (AD) is costly, time consuming and burdened by a very low success rate. An alternative strategy is drug repositioning, redirecting existing drugs for another disease. The large amount of biological data accumulated to date warrants a comprehensive investigation to better understand AD pathogenesis and facilitate the process of anti-AD drug repositioning. Hence, we generated a list of anti-AD protein targets by analyzing the most recent publically available 'omics' data, including genomics, epigenomics, proteomics and metabolomics data. The information related to AD pathogenesis was obtained from the OMIM and PubMed databases. Drug-target data was extracted from the DrugBank and Therapeutic Target Database. We generated a list of 524 AD-related proteins, 18 of which are targets for 75 existing drugs-novel candidates for repurposing as anti-AD treatments. We developed a ranking algorithm to prioritize the anti-AD targets, which revealed CD33 and MIF as the strongest candidates with seven existing drugs. We also found 7 drugs inhibiting a known anti-AD target (acetylcholinesterase) that may be repurposed for treating the cognitive symptoms of AD. The CAD protein and 8 proteins implicated by two 'omics' approaches (ABCA7, APOE, BIN1, PICALM, CELF1, INPP5D, SPON1, and SOD3) might also be promising targets for anti-AD drug development. Our systematic 'omics' mining suggested drugs with novel anti-AD indications, including drugs modulating the immune system or reducing neuroinflammation that are particularly promising for AD intervention. Furthermore, the list of 524 AD-related proteins could be useful not only as potential anti-AD targets but also considered for AD biomarker development.

Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, 60 Leonard Street, Toronto, Ontario, Canada M5T 2S8, Neurology I, Rita Levi Montalcini Department of Neuroscience, University of Torino, Torino, Italy, Regional Neurogenetic Centre, Lamezia Terme, Azienda Sanitaria Provinciale Catanzaro, Catanzaro, Italy, Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Florence, Italy, Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, Centro Dino

The G(4)C(2) repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G(4)C(2) repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5' of the G(4)C(2) repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD).