Despite its identification as a key checkpoint regulator of microglial activation in Alzheimer’s disease, the overarching role of CX3CR1 signaling in modulating mechanisms of Aβ driven neurodegeneration, including accumulation of hyperphosphorylated tau is not well understood.Cx3cr1/++ and 5xFAD;Cx3cr1−/− mice using immunohistochemistry, western blotting, transcriptomic and quantitative real time PCR analyses of purified microglia. Flow cytometry based, in-vivo Aβ uptake assays are used for characterization of the effects of CX3CR1-signaling on microglial phagocytosis and lysosomal acidification as indicators of clearance of methoxy-X-04+ fibrillar Aβ. Lastly, we use Y-maze testing to analyze the effects of Cx3cr1 deficiency on working memory.Accumulation of soluble and insoluble Aβ species, microglial activation, synaptic dysregulation, and neurodegeneration is investigated in 4- and 6-month old 5xFAD;Cx3cr1−/− mice is characterized by increased deposition of filamentous plaques that display defective microglial plaque engagement. Microglial Aβ phagocytosis and lysosomal acidification in 5xFAD;Cx3cr1−/− mice is impaired in-vivo. Interestingly, Cx3cr1 deficiency results in heighted accumulation of neurotoxic, oligomeric Aβ, along with severe neuritic dystrophy, preferential loss of post-synaptic densities, exacerbated tau pathology, neuronal loss and cognitive impairment. Transcriptomic analyses using cortical RNA, coupled with qRT-PCR using purified microglia from 6 month-old mice indicate dysregulated TGFβ-signaling and heightened ROS metabolism in 5xFAD;Cx3cr1−/− mice. Lastly, microglia in 6 month-old 5xFAD;Cx3cr1−/− mice express a ‘degenerative’ phenotype characterized by increased levels of Ccl2, Ccl5, Il-1β, Pten and Cybb along with reduced Tnf, Il-6 and Tgfβ1 mRNA.Disease progression in 5xFAD;Cx3cr1 deficiency impairs microglial uptake and degradation of fibrillar Aβ, thereby triggering increased accumulation of neurotoxic Aβ species. Furthermore, loss of Cx3cr1 results in microglial dysfunction typified by dampened TGFβ-signaling, increased oxidative stress responses and dysregulated pro-inflammatory activation. Our results indicate that Aβ-driven microglial dysfunction in Cx3cr1−/− mice aggravates tau hyperphosphorylation, neurodegeneration, synaptic dysregulation and impairs working memory.The online version contains supplementary material available at 10.1186/s13024-022-00545-9. CR1, CD33, MEF2C, HLA-DRB5-DRB1, PTK2b and TREM2 that significantly increase the risk of AD [Neuroinflammation and glial activation in Alzheimer’s disease (AD) precede the onset of extracellular β-amyloid (Aβ) plaque deposition and evolve during the development of neurofibrillary tangles (NFTs) and neurodegeneration. These underlying signaling pathways are postulated to lay the framework for cognitive impairment seen in AD . Single-Cx3cr1 deficiency is associated with a transient deficit in microglial abundance during early post-natal development [2+ influx into neurons, thereby protecting against neuronal excitotoxicity [Cx3cr1 increases pro-inflammatory microglial signaling that correlates with heightened loss of dopaminergic neurons in the substantia nigra in murine models of Parkinson’s disease [Cx3cr1 deficiency is associated with increased SOD1 aggregation, neuronal loss and inflammatory microglial activation with impaired autophagy-lysosomal degradation pathways and autophagosome maturation [Recently, loss-of-function variants in the microglial fractalkine receptor (CX3CR1) have been associated with worsened Braak staging in AD and neurodegeneration along with reduced survival in amyotrophic lateral sclerosis (ALS), implicating these SNPs as disease-modifying variants in neurodegenerative diseases –8. Signaelopment , 12. Thielopment , 12, reselopment . CX3CR1-toxicity –15. Inde disease , 17. Simturation , 19. TheCx3cr1 in plaque-associated microglia is a primary event in the neuroinflammatory cascade in neurodegenerative diseases [Cx3cr1 expression represents a shift in microglia towards a protective phenotype associated with increased expression of Trem2, Apoe, CD68, Axl, MerTK and Lpl, indicative of enhanced capacity for TREM2-dependent plaque compaction, Aβ phagocytosis and lipid metabolism [Cx3cr1 and P2ry12 and upregulation of pro-inflammatory markers like Clec7a, Ccl5, Ccl2, IL1b, and Nos2. These neurodegenerative microglia are associated with neuritic plaques, degenerating neurons and trigger a loss of tolerogenic responses in EAE and ALS [Cx3cr1 has distinct effects on plaque clearance and subsequent neurotoxicity in AD.Single-cell RNA sequencing (scRNA-seq) studies have suggested that downregulation of diseases –22. Attetabolism . In cont and ALS . Thus, dCx3cr1 have provided insights into the divergent role of fractalkine signaling in amyloidosis and tauopathy [Cx3cr1 deficiency results in a gene-dose dependent reduction in fibrillar Aβ (fAβ) burdens in early disease, suggesting that a loss of CX3CR1 is beneficial in Aβ pathogenesis. Interestingly, 4 month-old APPPS1;Cx3cr1−/− mice display impaired microglial plaque engulfment with altered activation of plaque-associated microglia as evidenced by reduced CD68 and CD45 immunoreactivity [Cx3cr1 enhances microglial activation, increases accumulation of Gallays+ neuronal NFTs and exacerbates cognitive dysfunction [Cx3cr1−/− mice drive the spread of pathological pTau, possibly via the IL-1β signaling pathway [Our previous studies using murine models of AD deficient in auopathy –25. In tfunction . Further pathway . OverallCx3cr1 deficiency exacerbates neurodegeneration and cognitive impairment with disease progression in 5xFAD animals by driving increased accumulation of neurotoxic oligomeric Aβ (oAβ), fibrillar Aβ (fAβ) plaques, intraneuronal inclusions of hyperphosphorylated tau (pTau) and enlarged foci of neuritic dystrophy. These effects are potentiated, in part, due to microglial dysfunction in 5xFAD;Cx3cr1−/− mice as evidenced by impaired microglial Aβ-phagocytosis and clearance as well as aberrant TGFβ-signaling, inflammatory activation and reactive oxygen species (ROS) metabolism. Lastly, in contrast to 5xFAD;Cx3cr1/++ mice in which pTau pathology strongly correlates with accumulation of compact plaques and small foci of neuritic dystrophy, aggravated deposition of pathological tau in the absence of Cx3cr1 is correlated with, oligomeric Aβ, filamentous Aβ and large dystrophic neurites.In this report, we investigate how microglial responses to Aβ shaped by CX3CR1 drive long term neurotoxicity in the 5xFAD model of amyloid disease. Our results indicate that ±). B6.129P2(Cg)-Cx3cr1tm1Litt/J mice on the congenic background were purchased through Jackson Laboratories . In these mice, the first 390 base-pairs of the coding exon 2 of the Cx3cr1 gene are replaced by the enhanced green fluorescent protein (EGFP) sequence, thereby resulting in a loss of Cx3cr1 gene expression. Trem2−/− mice on the C57BL/6 J background were purchased through Jackson Laboratories  and mated with 5xFAD± mice to generate 5xFAD;TREM2−/− mice. 5xFAD± mice were mated with Cx3cr1−/− animals, and progeny were subsequently intercrossed to generate 5xFAD±;Cx3cr1−/− cohorts . Pathology in the absence of Cx3cr1 was compared to age-matched 5xFAD±;Cx3cr1/++  mice throughout the study. Age-matched littermates that do not express 5xFAD mutations  were used as controls throughout. All animals were housed in animal facilities within Stark Neurosciences Research Institute (SNRI) at The Indiana University School of Medicine (IUSM), accredited by the Association and Accreditation of Laboratory Animal Care. Animals were maintained according to USDA standards and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments were approved by the IUSM Institutional Animal Care and Use Committee.5xFAD mice on the C57BL/6 J background were obtained through Jackson Laboratories in collaboration with the Model-AD Center at the IU School of Medicine . 5xFAD mice were maintained as hemizygotes for the APP and PSEN1 transgenes  solution, de-stained with 70% ethanol and washed with 1XPBS prior to being coverslipped with ProlongGold.Three Male and 3 female mice of each genotype were used for all histochemical analyses, based on power analyses for an 80% probability of detecting a 25% change in AD pathological outcomes, based on previous publications . Brain t2O2 in 1XPBS for 30 min at RT to quench endogenous peroxidases. Antigen retrieval was performed by incubating sections in 10 mM sodium citrate (pH = 6.00) at 90ºC for 10 min. Sections were incubated in blocking solution containing 5% normal goat serum, 0.5% Triton-X100 and Mouse-On-Mouse (M.O.M) blocking reagent (1:1000) for 1 h at RT. Tissues were incubated overnight at 4ºC in blocking buffer with Mouse α-AT8. Following incubation with biotinylated α-mouse IgG , slices were developed using the VECTASTAIN Elite ABC kit (Vector Laboratories) and DAB. Sections were mounted and coverslipped using Permount . Details for all primary antibodies used for immune-fluorescent and DAB staining are listed in Supplementary Table For 3,3′-diaminobenzidine (DAB) staining, sections were incubated in a solution of 1% H+ microglia in B6;Cx3cr1/++ and B6;Cx3cr1−/− mice was done using the ‘Skeletonize’ and ‘Analyze Skeleton’ plugins in ImageJ. For analysis of plaque circularity and Iba1 occupancy, 15–20 μm Z-stacks were imaged at a 60X magnification. Circularity analysis on ThioS+ plaques was done using the ‘Shape Descriptors’ plugin in ImageJ. Cut offs for plaque circularity were defined as previously published by Yuan P. et al. [+ plaques, regions-of-interest (ROIs) were traced along ThioS+ plaque borders in serial sections co-stained with ThioS and Iba1. Defined ROIs were applied to the Iba1 layer, and the percentage of area within the ROI positive for Iba1 immunoreactivity was quantified. Neuronal numbers within the subiculum were counted automatically using the particle analysis feature. The watershed plugin was used for segmentation of tightly packed NeuN+ cell bodies. Dystrophic neurites were counted manually. For size classification, the line selection tool was used to manually threshold out particles less than 500 µm (50-500 µm), followed by subtraction of the number of dystrophic neurites > 500 μm (500-1000 µm) from the total number of dystrophic neurites.High resolution fluorescence imaging was done using the Nikon AR1 confocal microscope. High-resolution, brightfield images were acquired using the CTR5000 upright Leica microscope. Post processing and analysis was done using ImageJ . Branching and junction analysis for Iba1. et al. , where fCx3cr1/++, B6;Cx3cr1−/−, 5xFAD;Cx3cr1/++and 5xFAD;Cx3cr1−/− were injected intraperitoneally (i.p.) with 10 mg/kg of methoxy-X04 and sacrificed 3 h post injection. Brain-derived mononuclear cells were prepared as previously described [+CD45low vs CD11b+CD45high profiles. To assess endolytic activation, cells were incubated with 20 nM Lysotracker™ DeepRed . Lysotracker™ DeepRed and methoxy-X04 were detected using the 633 nm and 421 nm lasers respectively. All experiments were done using appropriate single-colored, compensation controls to eliminate any non-specific bleed-through due to spectral overlap.fAβ internalized by resident microglia was analyzed using methoxy-X04, a brain-penetrant dye that specifically binds to fibrillar β-sheet deposits . Brieflyescribed . Percoll+ microglial cells were purified using the Sony MA900 cell sorter. FACS purified cell populations were recovered by centrifugation at 400 × g for 7 min at 4 °C, resuspended in TRIzol  and stored at − 80 °C for subsequent RNA extraction and cDNA synthesis. Cell yields ranged between 400,000–600,000 CD11b+ microglia per brain.For microglial purification, percoll-derived mononuclear cells, resuspended in FACS buffer were Fc-blocked with rat anti-mouse FcγIII/II MAb  and incubated with PE-conjugated α-CD11b (M1/20) antibodies. CD11bGapdh (for qRT-PCRs using FACS purified microglia) or Hprt  and expressed as fold changes relative to microglia isolated from 5xFAD;Cx3cr1/++ females (for qRT-PCRs using FACS purified microglia) or B6;Cx3cr1/++ mice  following the 2−ΔΔCt method, where ΔΔCt = ΔCt (Target sample) – ΔCt (Reference sample). Taqman probes used are as follows – Microglial activation: Purinergic receptor P2Y12 (P2ry12: Mm01289605_m1), Itgam (Cd11b: Mm00434455_m1), Ionized calcium binding adaptor molecule-1 (Iba1: Mm00479862_g1); Spi1/Pu.1: Mm00488140_m1, Apolipoprotein E , C-Type Lectin Domain Containing 7A , Cystatin F , transforming growth factor β-1 , transforming growth factor-β-receptor 1 . Neuroinflammation: chemokine (C–C motif) ligand 2 , chemokine (C–C motif) ligand 5 , interleukin 1β , interleukin 6 , tumor necrosis factor . ROS metabolism: phosphatase and tensin homolog , PTEN-induced kinase-1 , cytochrome b-245 beta chain .150-200µls of homogenized cortical lysates were mixed with an equal volume of QIAzol lysis reagent (QIAGEN RNeasy kits). RNA was isolated using QIAGEN RNeasy kit as per the manufacturer’s protocol and quantified using NanoDrop (Thermo Scientific). 1 µg RNA was used to synthesize cDNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). Cellular TRIzol fractions were treated with chloroform, and RNA was precipitated with 2-propanol, washed with 75% ethanol and resuspended in RNAse free water. Following DNAse treatment using the DNA Free™ kit (Ambion), cDNA was synthesized using the Moloney murine leukemia virus (MMLV) reverse transcriptase kit (Invitrogen). Gene expression was normalized to Cx3cr1−/− mice with respect to 5xFAD;Cx3cr1/++ cohorts were done using the Panther Classification System (http://www.pantherdb.org/). KEGG enrichment analyses for determination of top signaling pathways altered in 5xFAD;Cx3cr1−/− mice were done using the VolcanoR software (http://45.8.90.25:3838/volcanoR/). Statistical differences for this data set were calculated using the Benjamini-Hotchberg method within nSolver, Panther Classification System and VolcanoR software.The NanoString Mouse Neuroinflammation gene expression panel (Cat # LBL-10492–02) was used for gene expression profiling on the nCounter platform  as described by the manufacturer. nSolver software was used for analysis of NanoString gene expression values. Normalization to identify differentially expressed genes (DEGs) was done by dividing counts within a lane by geometric mean of the housekeeping genes from the same lane. Gene Ontology analyses for determination of top functional pathways affected in 5xFAD;Cx3cr1/++ and 5xFAD;Cx3cr1−/− mice. Supplemental Fig. Cx3cr1/++ and B6;Cx3cr1−/− mice. Supplemental Fig. Cx3cr1 deficiency does not alter microglial proliferation, microglial recruitment to Aβ plaques and TREM2 levels in the 5xFAD brain. Supplemental Fig. Cx3cr1/++ vs. 5xFAD;Cx3cr1−/− mice. Supplemental Fig. Cx3cr1/++ and B6;Cx3cr1−/− mice. Supplemental Fig. + microglial subsets in B6 and 5xFAD mice. Supplemental Fig. Cx3cr1/++ and 5xFAD;Cx3cr1−/− mice. Supplemental Fig. Cx3cr1−/− mice. Supplemental Files Cx3cr1−/− vs. 5xFAD;Cx3cr1/++ mice.Supplemental Table Cx3cr1−/− mice between post-natal days 8 through 28 [FACS purified, CD11b+CD45+ microglia from 2 month-old B6;Cx3cr1−/− mice are not evident in aged microglia from 12 and 24 month-old Cx3cr1 deficient mice as compared to cells isolated from age-matched B6;Cx3cr1/++ animals [Cx3cr1 deficiency has transient and subtle effects on the transcriptional landscape of homeostatic microglia. To ascertain that no overt microglial defects persist into adulthood, we examined 6 month- old B6;Cx3cr1/++ and B6;Cx3cr1−/− mice for microglial abundance and homeostatic activation. qRT-PCR analyses using cortical mRNA revealed similar expression of canonical microglial genes namely Pu.1, Iba1, P2ry12 and Cd11b  to visualize fAβ plaques. ThioS+ plaques deposited in 5xFAD;Cx3cr1−/− mice appeared significantly more diffuse when compared to those in 5xFAD;Cx3cr1/++ mice  along with soluble, oligomeric Aβ (oAβ) are associated with neurotoxicity in AD –34. To aCx3cr1−/− mice correlates with dysregulation of plaque associated microglial responses, we used flow-cytometry and histology to visualize microglia associated with fAβ plaques. Consistent with increased plaque burdens  and pro-survival signaling (Bcl2l1) in 5xFAD;Cx3cr1−/− mice when compared to Cx3cr1/++ counterparts , along with increased Cst7, P2ry17 and Tgfbr1 levels in 5xFAD;Cx3cr1−/− mice   as compared to age-matched 5xFAD;Cx3cr1/++ cohorts (~ 36%), corresponding to a mild, but significant increase in the fAβ+ CD11b+ CD45high microglial subset in these animals . In contrast, significant correlation was observed between cortical pTau and intermediate fAβ  and diffuse fAβ  in 5xFAD;Cx3cr1−/− animals.To identify mechanisms of overt neuronal and synaptic loss, we investigated the accumulation of pTau in 5xFAD mice. 6 month-old 5xFAD mice showed increased pTau pathology as compared to age-matched B6 controls . CX3CL1-ct is in-turn cleaved by γ-secretase to release the intracellular domain of CX3CL1 (-ICD). Using transgenic 5xFAD mice that overexpress CX3CL1-ct , nal loss , 41. Laspus Fig.  in the aCx3cr1 deficiency aggravates the accumulation of ‘dark microglia’. These cells display signs of oxidative stress, including an electron-dense cytoplasm and nucleoplasm, cytoplasmic fragmentation, dilation of Golgi- and ER membranes and mitochondrial alterations [Cx3cr1−/− microglia show downregulation of homeostatic markers like P2RY12 and display extensive engulfment of presynaptic axon terminals and postsynaptic dendritic branches. Similar ‘dark’ microglia associated with Aβ plaques in APPPS1 mice [Cx3cr1−/− mice  an increased recruitment of signaling proteins to pre-synaptic terminals may be reflective of compensation for the lack of synaptic coupling/plasticity observed in our mice  oAβ  active elimination of neurons by neurodegenerative microglia may be dictated by the quality of intraneuronal pathological protein aggregates and b) microglial neurodegenerative responses modulated by CX3CR1 may be subject to region-dependent microglial heterogeneity along with regional differences in neuronal susceptibility , 52, 53.in-vivo link between CX3CR1-dependent microglial activation, aberrant homeostasis of soluble and insoluble Aβ and subsequent effects on chronic neurodegeneration in AD. This work suggests that the long term neurodegenerative changes including accumulation of pathological tau, synaptic dysregulation and altered neuronal homeostasis in the absence of Cx3cr1 correlate with the quality along with the abundance of extracellular Aβ. We hypothesize that Cx3cr1 deficiency in AD impairs microglial endolytic activation, thereby affecting uptake and degradation of fibrillar Aβ, and triggering an accumulation of neurotoxic Aβ species. Lastly, the neurotoxic Aβ microenvironment in the absence of Cx3cr1 further drives increased microglial dysfunction typified by aberrant inflammatory activation, ROS metabolism and a skewing of the microglial response to a neurodegenerative phenotype.Clinical outcomes in AD are a net sum of a range of pathological microglial activation states that are dynamically controlled by CX3CR1-signaling in response to the diverse pathological features of AD. Thus, CX3CR1 may simultaneously mediate distinct microglial phenotypes based on contextual cues received via their interaction with Aβ, dysfunctional synapses, dystrophic neurites or distressed neurons within their microenvironment. While our understanding of the overarching role of microglial CX3CR1 on disease mechanisms that affect neuronal and synaptic health is still primitive, this study is the first to provide an Additional file 1: Supplemental Fig. 1. Cx3cr1 deficiency does not alter microglia in 6 month-old B6 mice. (A) Quantitative real time PCR (qRT-PCR) based quantification for cortical expression of microglial genes (i) Pu.1, (ii) Iba1, (iii) P2ry12 and (iv) Cd11b in B6;Cx3cr1+/+ (black bars) and B6;Cx3cr1-/- (grey bars) mice. Data represents mean ddCT values for n = 8  mice for each genotype. (B) Flow cytometry based quantitation of CD11b+ microglia in 6 month-old B6;Cx3cr1+/+ (black bars) and B6;Cx3cr1-/- (grey bars) mice. Flow-cytometry plots representative of n = 8 mice  for each genotype. (C) Representative, high-resolution confocal microscopy images of Iba1+ microglia from 6 month-old B6;Cx3cr1+/+ and B6;Cx3cr1-/- mice used to quantify number of branches / cell, and junctions / cell. Branching and junctions quantified for a total of 72 microglia selected from n = 6  for each genotype. Statistical analysis done using two-tailed, standard Student’s t-test with Welch’s corrections for unequal SDs. *p = 0.02.Additional file 2: Supplemental Fig. 2.Cx3cr1 deficiency does not impair plaque associated microglial proliferation and microglial recruitment to Aβ plaques. (A-i) Representative flow-cytometry plots showing increased (A-ii) proportion and (A-iii) numbers of CD11b+ microglia in 6 month-old 5xFAD;Cx3cr1+/+ (black bars) and 5xFAD;Cx3cr1-/- (grey bars) mice. Data represents mean microglial proportions / numbers quantified using n =6  mice for each genotype. Error bars represent SEM. Quantification of (B-i) %Iba1+ areas and (B-ii) %Iba1+ areas normalized to %ThioS+ areas in the cortices of 6 month-old 5xFAD;Cx3cr1+/+ (black bars) and 5xFAD;Cx3cr1-/- (grey bars) mice. Error bars represent SEM. (C) Representative images of Ki67+Iba1+DapI+ proliferating microglia associated with diffuse ThioS+ plaques in 5xFAD;Cx3cr1+/+ (top panels) and 5xFAD;Cx3cr1-/- (bottom panels) mice. Scale bars = 10µm. (D) Quantification of the proportion of ThioS+ plaques associated with proliferating microglia based on colocalization of Ki67, Pu.1 and Iba1 in cortices of 5xFAD;Cx3cr1+/+ (black bar) and 5xFAD;Cx3cr1-/- (grey bar). (E) Quantification of proportions of Ki67+Iba1+DapI+ microglia associated with cortical, ThioS+ plaques in 6 month-old 5xFAD;Cx3cr1+/+ (black bars) and 5xFAD;Cx3cr1-/- (grey bars) mice. Data in C-E processed using n = 6  of each genotype. Data in D, E based on analysis of 10,000 – 15,000 individual microglia associated with 250-300 plaques per genotype. (F) Quantitative real time PCR (qRT-PCR) analyses of Trem2 and Tyrobp expression in CD11b+ microglia purified from n = 8  5xFAD;Cx3cr1+/+ and 5xFAD;Cx3cr1-/- mice. (G) Quantitation of TREM2 protein levels in cortical lysates of 6 month-old 5xFAD;Cx3cr1+/+ (black bar) and 5xFAD;Cx3cr1-/- (grey bar) mice. Lysates from age-matched 5xFAD;Trem2-/- mice were used as negative controls. Data represents mean TREM2 concentrations in lysates from 6 mice  of each genotype. Error bars represent SEM. Statistics in A, B and C calculated using two-tailed, standard Student’s t-test with Welch’s correction for unequal SDs. **p < 0.005, ***p = 0.002.Additional file 3: Supplemental Fig. 3. Top biological processes and signaling pathways altered in 6 month-old 5xFAD;Cx3cr1-/- mice when compared to 5xFAD;Cx3cr1+/+ mice. RNA extracts from cortical lysates from n = 6 animals  were used for Nanostring based transcriptional profiling. (A) Gene ontology (GO) analyses showing the top 11 altered biological pathways in female vs. male 5xFAD;Cx3cr1-/- mice with respect to sex-matched 5xFAD;Cx3cr1+/+ mice. Venn diagram highlighting (B) common biological pathways using GO analysis and (C) common neuropathology signaling pathways and cellular processes using KEGG analysis in male and female 5xFAD;Cx3cr1-/- mice as compared to 5xFAD;Cx3cr1+/+ mice. GO and KEGG analysis done on significant differentially expressed genes (DEGs), which were calculated using the Benjamini-Hochberg test in the Nanostring nCounter Analysis Software.Additional file 4: Supplemental Fig. 4. Microglial signatures, inflammatory activation and oxidative signaling are not altered by Cx3cr1 deficiency in 6 month-old B6 mice. cDNA synthesized using cortical RNA from 6 month-old B6;Cx3cr1+/+ (black bars) and B6;Cx3cr1-/- (grey bars) mice was used to perform qRT-PCR analyses. (A) Quantification of microglial activation phenotype assessed by gene-expression for (i) Trem2, (ii) Clec7a, (iii) Cst7, (iv) Apoe, (v) Tgfβ1, (vi) Tgfβr1, (vii) Axl and (viii) Mertk. (B) Inflammatory activation assessed by gene expression for (i) Ccl2, (ii) Ccl5, (iii) Il-1β, (iv) Il-6 and (v) Tnf. (C) Quantification of oxidative stress signaling assessed by gene expression for (i) Pten, (ii) Cybb, (iii) Pink-1. Data represents mean ddCT values for n = 8 mice  for each genotype. All data normalized to B6;Cx3cr1+/+ mice. Statistical analysis done using two-tailed, standard Student’s t-test with Welch’s corrections for unequal SDs. **p = 0.003.Additional file 5: Supplemental Fig. 5. Flow cytometry based analysis of ex-vivo, microglial phagocytosis of fibrillar Aβ (fAβ). (A) Gating strategy to identify CD11b+ CD45low vs. CD11b+ CD45high microglia in methoxy-X04 injected 6 month-old cohorts. Flow cytometry plots shown represent data from B6;Cx3cr1+/+ and 5xFAD;Cx3cr1+/+ mice. (B) Flow cytometry plots showing the absence of non-specific retention of methoxy-X04 in CD11b+ microglia in B6;Cx3cr1+/+ mice and GFP+ microglia in B6;Cx3cr1-/- mice. (C) Flow cytometry data showing proportions of methoxy-X04+ microglia within CD11b+ CD45low and CD11b+ CD45high subpopulations in 6 month-old 5xFAD;Cx3cr1+/+ mice. Microglia identified using gating strategy showed in (A). fAβ+ microglia are further classified into methoxy-X04low / fAβlow and methoxy-X04high / fAβhigh sub-populations. (D) Flow cytometry data showing identification of microglia in 5xFAD;Cx3cr1-/- mice based on CD11b+ vs. GFP+ expression for analysis of proportion of phagocytic, methoxy-X04+ sub-populations. Data in B and D show that the use of CD11b vs. GFP does not alter the profiles of fAβ+ microglia, and there is no non-specific, spectral overlap between CD11b, GFP and methoxy-X04 channels. All data representative of flow-cytometry analyses done using n = 5 female and 5 male mice of each genotype and processed in a single experiment. All experiments done using appropriate single-colored compensation controls to eliminate spectral overlap.Additional file 6: Supplemental Fig. 6. Microglial fAβ uptake and endolytic activation is altered in 4 month-old 5xFAD;Cx3cr1-/- mice. (A) Representative flow cytometry data showing methoxy-X04+/ fAβ+ CD11b+ microglia, and quantification of %Methoxy-X04+ cells within CD11b+ CD45low and CD11b+ CD45high microglial sub-populations in 5xFAD;Cx3cr1+/+ (black bars) and 5xFAD;Cx3cr1-/- (grey bars) mice. Quantification of mean fluorescence intensities (MFI) of Lysotracker-DR for Methoxy-X04low and Methoxy-X04high microglia within (B) CD11b+ CD45low and (C) CD11b+ CD45high sub-populations in 5xFAD;Cx3cr1+/+ (black bars) and 5xFAD; Cx3cr1-/- (grey bars) mice. All data is analyzed using n = 4 females for each genotype. Statistical analysis done using Two-way ANOVA followed by Tukey’s post-hoc tests.Additional file 7: Supplemental Fig. 7. Larger dystrophic neurites (DNs) in 6 month-old 5xFAD;Cx3cr1-/- mice. Representative, high-resolution confocal images for (A) LAMP1+ DNs and (B) Ubiquitin+ DNs associated with ThioS+ plaques in 6-month old 5xFAD;Cx3cr1+/+ and 5xFAD;Cx3cr1-/- mice. Quantification of DNs in 6 month-old 5xFAD;Cx3cr1+/+ (black bars) and 5xFAD;Cx3cr1-/- (grey bars) using (A) α-LAMP1, (B) α-Ubiquitin and (C) α-nT-APP antibodies. Dystrophic neurites were quantified on the basis of their size, defined as small  vs. large . Data in C-E represents mean cortical dystrophic neurite abundance calculated using multiple sections from n = 6 mice . Statistical analysis done using Two way ANOVA  followed by Tukey’s post-hoc tests. *p <0.05, **p < 0.005, ns – non-significant.Additional file 8: Supplemental Table 1.Additional file 9: Supplemental Table 2. Pearson’s correlation analyses between cortical pTau pathology, small vs. large dystrophic neurites and plaque diffusivity in 6 month-old 5xFAD; Cx3cr1+/+ and 5xFAD; Cx3cr1-/- mice. Pearson’s correlation analyses were done using data from Supplemental Figure + cortical areas) to investigate interactions between small (<500µm) and large (>500µm) (A) Ubiquitin+, (B) nT-APP+ and (C) LAMP1+ dystrophic neurites and their relation to compact vs. intermediate vs. diffuse plaques and accumulation of AT8+ pTau in the cortices of 6 month-old 5xFAD; Cx3cr1+/+ and 5xFAD; Cx3cr1-/- mice. All data calculated using n = 6  of each genotype as described in materials and methods and related figure legends. Correlation matrices were calculated using imageJ. All data was tested for normality using the Anderson-Darling Test, and the Sharpiro-Wilk test for normality distribution. Significances were calculated using standard two-tailed t-tests with a 95% confidence interval. All significant interactions have been indicated in red. *p = 0.01, **p < 0.001, ns = not significant.Additional file 10. Supplemental Materials and methods.


Neurofibrillary tangle (NFT) accumulation in the entorhinal cortex (EC) precedes the transformation from cognitive controls to mild cognitive impairment and Alzheimer’s disease (AD). While tauopathy has been described in the EC before, the order and degree to which the individual subfields within the EC are engulfed by NFTs in aging and the preclinical AD stage is unknown.We aimed to investigate substructures within the EC to map the populations of cortical neurons most vulnerable to tau pathology in aging and the preclinical AD stage.within the eight entorhinal subfields (described by Insausti and colleagues) at the preclinical stages of AD. We validated with immunohistochemistry and labeled the NFT density ratings on ex vivo MRIs. We measured subfield cortical thickness and reconstructed the labels as three-dimensional isosurfaces, resulting in anatomically comprehensive, histopathologically validated tau “heat maps.”We characterized phosphorylated tau (CP13) in 10 cases at eight well-defined anterior-posterior levels and assessed NFT density p < 0.001). We report an age-dependent anatomically-specific vulnerability, with all cases showing posterior tau pathology, yet older individuals displaying an additional anterior tau burden. Finally, cortical thickness of each subfield negatively correlated with respective tau scores (p < 0.05).We found the lateral EC subfields ELc, ECL, and ECs  to have the highest tau density in semi-quantitative scores and quantitative measurements. We observed significant stepwise higher tau from anterior to posterior levels (Our findings indicate that posterior-lateral subfields within the EC are the most vulnerable to early NFTs and atrophy in aging and preclinical AD. The entorhinal cortex (EC) functions as the main gateway for cortical information to the hippocampus . Abnormain vivo and ex vivo neuroimaging approaches in understanding the substructure of the EC. A high resolution functional magnetic resonance imaging (fMRI) report identified functional subdivisions and connectivity between the perirhinal cortex and the anterior-lateral EC, and between the parahippocampal cortex and the posterior-medial EC in the human brain [Some advances have been made using an brain . Anotheran brain . In addian brain . Animal an brain , while san brain . Anatomian brain . Still, ex vivo MRI investigations have undertaken three-dimensional mappings of NFTs in the medial temporal lobe regions including the entorhinal, hippocampus, amygdala, and temporal pole areas [ex vivo reports, however, have lacked entorhinal subdivision specificity and histopathology validation approaches. And, of course, in vivo studies fail to provide this specificity as well due to poor spatial resolution and lack of ground truth tissue samples.Pathologic diagnosis relies on two-dimensional data, and as such, the bulk of AD histology studies have been limited to just a few sections in the medial temporal lobe. Thus, the three-dimensional axes have been vastly understudied regarding NFT formation and have lacked subfield specificity, which could pose functional implications for early disease stages. Initial le areas . Based ole areas . Previouwithin the EC subfields and at well-defined anterior-posterior anatomical levels. We focused exclusively on the preclinical stages of AD, the first two Braak and Braak stages [ex vivo MRIs with corresponding cortical thickness measures on all cases. The combination of methods—ex vivo MRI and histology—provide validation and allow for application to neuroimaging and will be useful for future in vivo imaging hypotheses. These descriptive maps bring together at least three factors of vulnerability: age, anatomical location, and phosphorylated tau immunoreactivity, showing the entorhinal tau vulnerability in 3D at the preclinical stages.To address these issues, we characterized phosphorylated tau and specifically assessed NFT density k stages . This chn = 3 right, n = 7 left) received from the Department of Neuropathology, Autopsy Suite at Massachusetts General Hospital . All samples were collected in accordance with the internal review board of Massachusetts General Hospital. All experiments met ethical standards and were approved by the internal review board of Massachusetts General Hospital. The samples comprised five males, four females, and one case with demographic information unavailable. Ages ranged from 59–84 years, 69.67±9.94; mean±std , a significant neurodegenerative marker [Based on clinical history, none of the cases had a diagnosis of Alzheimer’s disease (AD) nor any other neurological diseases. We followed the NIA Alzheimer’s Association guidance  to contre marker  that impe marker . All demμm isotropic providing full Fourier encoding [μm [ex vivo tissue to visualize and distinguish microanatomy in MRI [Cases were scanned in a whole body high-field 7T Siemens Magnetom . Two radio frequency coils were used: a 4-turn solenoid coil, giving a resolution of 100encoding , and theding [μm . Medial y in MRI . Three Mμm, collected serially, and frozen at –20°C in cryoprotectant solution. During sectioning, blockface photographs were collected serially using a Canon EOS-1D Mark IV camera  with a compatible LED ring flash mounted above the microtome. Tissue for Nissl staining was selected at approximately every 10 sections and thus separated by 500μm. Tissue sections were hand mounted onto glass slides, dried overnight, and stained with thionin for Nissl substance. For the Nissl staining protocol, slides were defatted in a chloroform and 100% ethanol solution, followed by pretreatment in an acetic acid, acetone, 100% ethanol and water (1:1:1:1) mixture, and stained in 8% buffered thionin for 5 min. Then, the slides were differentiated in 70% ethanol with 5–10 drops of glacial acetic acid to the ideal contrast before they were dehydrated in a series of ethanol solutions with increasing concentration, cleared in xylene, and finally coverslipped with Permount .Temporal lobe blocks were cryoprotected in a 20% glycerol/2% dimethyl sulfoxide solution for two weeks. Tissue blocks were sectioned coronally on a freezing sliding microtome  at 50α-synuclein; goat anti-rabbit for TDP-43) were diluted in 1.5% NGS in PBS at 1:200 and incubated for one hour at room temperature . Tissue was subsequently washed with PBS and incubated with an ABC kit  for 30 min at room temperature. To visualize the staining, tissue was incubated with chromogen 3’3-diaminobenzidine  in a solution of 0.003% 3’3-diaminobenzidine and 0.3% hydrogen peroxide for 10 min. Tissue was rinsed three times with PBS prior to mounting on slides and air dried overnight. Immunostained sections were dehydrated in a series of ethanol solutions with increasing concentrations, cleared with xylene, and coverslipped with permount . Negative control sections were processed with the identical procedure, but the primary antibody was omitted. All experiments were run with a positive control tissue section.Immunostaining was performed on free-floating sections in 12-well plates. Adjacent sections to Nissl-stained sections were selected for immunostaining. Sectioned tissue was washed three times in phosphate buffer saline (PBS) to rid it of cryoprotectant glycerol solution. Sections were incubated with 0.5% Triton X-100 in 3% hydrogen peroxide for 20 min to quench endogenous peroxide, then rinsed in PBS three times for 5 min each. The tissue was blocked using 5% nonfat dry milk in PBS for 2 h at room temperature and rinsed in PBS. All primary antibodies  were diln = 6), the NFTs, pretangles, and neuropil threads (NTs) were confined to the perirhinal cortex area 35a (Braak and Braak stage I) and entorhinal cortex (Braak and Braak stage II) [n = 4) displayed isolated or small numbers of NFTs and/or NTs in the hippocampal subfields CA1 and CA2. These hippocampal NFTs, however, were not band-like at CA1, nor was CA2 compromised with large and strongly stained NFTs, excluding them from a stage III classification [Tissue sections immunostained for CP13 were assessed for AD staging and severity by three raters . CP13 stains for hyperphosphorylated tau both at the pretangle state, in which neurons display nonfibrillar punctate regions, and at the later stage in which they form intra- and extra-neuronal fibrillary tangles . It has tage II) . A subsefication . All casfication . All casfication . Case 10fication . We seleWe parcellated the subfields of the EC in Nissl-stained sections according to Insausti et al., 1995 using a Nikon SMZ1000 microscope . The eight entorhinal subfields parcellated were the olfactory subfield (EO), which is the most anterior and located in the medial parahippocampal gyrus, the rostral subfield (ER) located anteriorly and adjacent to EO, the lateral rostral subfield (ELr) found anteriorly near the collateral sulcus, the medial intermediate subfield (EMI) situated in the gyrus ambiens, the intermediate subfield (EI), which is positioned between the gyrus ambiens and the collateral sulcus, the lateral caudal subfield (ELc), which is an intermediate-posterior subdivision located long the posterior opening of the collateral sulcus, the caudal subfield (ECs) located posteriorly on the crown of the anterior parahippocampal gyrus, and finally the caudal limiting subfield (ECL), which is the most posterior subdivision in the entorhinal cortex, and is located most posterior on the crown of the anterior parahippocampal gyrus. We used the semiannularis sulcus as a medial landmark for EO and EMI see c, d. NotEight levels were established to investigate tau density in the anterior-posterior axis. We used three main criteria to establish uniform anterior-posterior levels: representation of all EC subfields, the presence of major anatomical landmarks in the surrounding hippocampal and amygdala regions, and consistent spacing between the levels themselves. The first and most important criterion defines the cytoarchitectural definition of each subfield . The secDensities of tau burden ) on individual EC subfields were semi-quantitatively scored using a rating scale which builds upon Braak and Braak’s well established rating scale  by focus* standard deviation). Finally, the number of tau positive pixels  and tau negative pixels  were extracted and used to calculate the tau ratio (tau ratio = number of tau positive pixels / number of tau negative pixels) as a quantitative measurement of region-specific tau burden.In addition to inter-rater tests performed to confirm reliability in the semi-quantitative protocol, we further validated our scores by employing a quantitative method that provided a supplementary reading of tau burden in these preclinical cases. The quantitative tau analysis was conducted using ImageJ/Fiji v.1.53c  and a seex vivo imaging (7 T) measuring cortical thickness, volume and surface area, and validated to Nissl staining [https://surfer.nmr.mgh.harvard.edu/). This manual labeling provides the ability to reconstruct tau density in a three-dimensional volume.Using cytoarchitectural features within the EC and the surrounding structures, we manually matched the coronal MRI slices with immunostained tau sections and Nissl-stained sections. staining . The semex vivo MRI scan, at the midpoint of its anterior-posterior length, and the last instance the subfield is present. Three equidistant medial-lateral locations were collected on each measured slide. Nine manual cortical thickness measurements were collected per subfield and per case, totaling 810 measurements. The multiple measurements per subfield were done to account for the changing anatomy across the medial-to-lateral and the anterior-posterior axes. Measurements for the subfield EMI were intentionally excluded due to the deformation from the test tube during the ex vivo MRI scans. Thickness measurements were averaged and correlated with mean CP13 semi-quantitative scores for each subfield. See Cortical thickness for each entorhinal subfield and perirhinal cortex was manually measured as the Euclidian distance from the pial surface of each subfield to the gray/white matter boundary using the measure function in Freeview. Three different slides were chosen for each subfield: when the subfield first appears in the ρ) were used to evaluate semi-quantitative and quantitative measurements for entorhinal burden across anterior-posterior levels. Spearman’s correlation tests were also used to determine agreement between semi-quantitative and quantitative measurements and to test the relationship between average CP13 scores and cortical thickness of each subfield. Pearson’s R was used to evaluate correlations between age and anterior pathology levels. Mann-Whitney U was used for nonparametric comparisons between semi-quantitative tau found in lateral and medial bifurcations of the EI and ECs subfields, as well as perirhinal tau. Cohen’s Kappa was used to assess inter-rater reliability on semi-quantitative tau burden scores. Kruskal Wallis and Dunn’s tests for post-hoc analyses were applied to determine statistical group differences in semi-quantitative as well as quantitative measurements across anterior-posterior levels and across subdivisions. Kruskal Wallis and Dunn’s tests were employed to assess statistical group differences in entorhinal and perirhinal cortical thickness measurements. All statistics used an alpha level of p < 0.05 as the threshold for significance, and were performed using Prism  and R-studio v.1.4.1 (The RStudio Team).Non-parametric correlations (Spearman’s Lat and EILat) and medial components (ECsMed and EIMed); these subfields indicate crucial transition regions from anterior to posterior portions of the EC and accordingly illustrate the most salient pathology gradient medially to laterally. We found that the lateral regions consistently displayed heavier pathology than their medial counterparts. Lat displaying more substantial tau pathology than ECsMed. Overall, tau pathology found in the ECsLat was significantly heavier than that in the ECsMed  and observed the most severe and least variable (std = 0.503) tau burden at level seven  and particularly found that three posterior-lateral entorhinal cortex subfields: ELc, ECL, and ECsLat had the heaviest tau burden density in semi-quantitative measures  = 25.56, el seven a. Level measures b.R = 0.6867, p < 0.05). One case .Overall, perirhinal cortex (subdivisions 35a and 35b) showed the highest tau density and was most severe at level seven . Mean seWe assessed the semi-quantitative tau pathology scores with an inter-rater reliability test. Two raters  evaluated the density of CP13-positive structures on each entorhinal and perirhinal cortices at every level in all 10 cases. Applying a Cohen’s Kappa test, we found substantial agreement among the two raters .ρ=0.775, p < 0.01; ρ=0.9048, p < 0.01; LAT subfields showing the highest CP13 tau burden  pathology in entorhinal subfields and perirhinal cortex. We found a strong positive correlation between semi-quantitative tau pathology scores and quantitative measurements  onto the reconciled MRI slides . The samThe reconstructed isosurfaces of the MRI labels highlight the most vulnerable segments in the tau burden severity map—the lateral/posterior portions of the EC—along with the individual anatomical variability for each case . ComplemMed = 3.24±0.35 mm, EILat = 3.31±0.23 mm, ELc = 3.10±0.31 mm, ECsMed = 2.80±0.52 mm, ECsLat = 2.68±0.21 mm, ECL = 2.78±0.20 mm. p < 0.0001) and perirhinal subdivisions  = 16.83, p < 0.01), along with a significant negative correlation between average entorhinal subfield cortical thickness measures and average entorhinal subfield CP13 tau scores .Lat, ECL, and ELc showed the highest tau density scores across both semi-quantitative scores and quantitative measurements. We segmented the boundaries of the entorhinal subfields based on phosphorylated tau staining; the entorhinal subfields EI and ECs were bifurcated and contained heavier pathology in lateral regions compared to medial ones. In addition, we observed age-dependent anatomically specific vulnerabilities in cases with similar Braak and Braak stages. The youngest-old cases (59–74 years) displayed NFTs in posterior regions of the EC, contrasting middle-old (75–84 years) cases which showed additional CP13-tau inclusions in the anterior EC; only one case indicated exception to this pattern. Our cortical thickness measurements using ex vivo MRI negatively correlated with tau density scores of their corresponding entorhinal subfield, providing tissue validation to a neuroimaging measure. We mapped our CP13 semi-quantitative scores onto the corresponding ex vivo MRI scans . Subfields ECsRI scans , and subRI scans . The reswithin the EC subfields and perirhinal cortex  . The latn ECsMed h. EILat subfield . Our ressubfield . Moreovecontrols . The finLAT, ECL, and ELc had the highest CP13 tau semi-quantitative scores  cases, contrasting our younger  cases , 6. WherAnterograde and retrograde tracing studies in the non-human primate have elucidated crucial connectivity patterns regarding the subfields within the EC. Posterior subfields EI, ELc, ECs, and ECL receive input from the retrosplenial cortex, a spatial and episodic memory region shown to display early pathologic changes in AD progression . Studiesex vivo scans, and the well-defined and trackable anterior-posterior levels which, as shown here, have varying degrees of vulnerabilities to NFTs . Labeled colors reflect corresponding semi-quantitative tau burden scores from Click here for additional data file.


Aberrant accumulation of β-catenin in the cell nucleus as a result of deregulation of the Wnt/β-catenin pathway is found in various types of cancer. Direct β-catenin targeting agents are being researched despite obstacles; however, specific β-catenin drugs for clinical treatments have not been approved so far. We focused on direct β-catenin targeting of potential therapeutic value as anticancer agents. This review provides recent advances on small molecule β-catenin agents. Structure-activity relationships and biological activities of reported inhibitors are discussed. This work provides useful knowledge in the discovery of β-catenin agents. Cyclin-D1, resulting in the initiation and progression of various types of carcinoma, including colon cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer and ovarian cancer -thymidine incorporation assay. Compounds 3 and 4, but also the non-halogenated derivative, 5, demonstrated activity two-fold superior to compound 1; other SAR modifications did not provide significant improvement over 1. The authors hypothesized the involvement of a variety of targets and observed partial agreement between the [3H]-thymidine incorporation and the TOPFlash assays: for example, compound 3 was superior to 1 in the [3H]-thymidine incorporation assay but showed weak activity in the TOPFlash assay; on the contrary, methyl benzoate 6 was weakly active in the [3H]-thymidine incorporation assay but was comparable to 1 in the TOPFlash assay.Kril and co-workers conducted structure-activity relationship (SAR) studies of patterns . Replaci7) as an inhibitor of the Wnt/β-catenin signaling pathway [7 inhibited Tcf luciferase reporter activity in HCT116 cells with luciferase-based reporter containing Tcf binding sites (TOP-Luc), while it showed little effect on FOP-Luc. Compound 7 decreased the cell viability of Wnt-dependent HCT116, HT115 and H23 cells and inhibited tumor growth in a xenograft mice model after 2 weeks’ treatment at 10–20 mg/kg. In these studies, 7 proved to bind to β-catenin in a concentration-dependent manner, promoted its degradation in a proteasome-dependent manner, and specifically downregulated Wnt/β-catenin target genes in DLD-1, SW480 and LS174T cancer cell lines. It was suggested as a potential therapeutic agent for the treatment of tumors dependant on the Wnt/β-catenin signaling pathway.High-throughput screening was conducted by Hwang’s group to identify inhibitors capable of modulating β-catenin-mediated Wnt signaling in HCT116 cancer cells. The authors conducted the HTS utilizing HCT116 cells with a mutant allele of β-catenin lacking Ser45, the phosphorylation target of CK1α, thus leading to high levels of active b-catenin. These studies led them to identify MSAB . Activation of c-Myc oncoprotein is one of the leading events of tumorigenesis and a high level of c-Myc correlates with the tumor’s survival. Direct or indirect suppression of c-Myc may slow down or reverse the cancer growth rate [9 was shown to have potential for the development of new anticancer therapies  showed IC50 of 1.65 and 1.82 μM, respectively, and was predicted with good plasma membrane permeability by Lipinski’s rule [11, bearing a benzyl group, exhibited inhibitory activity comparable to 10. Compound 10 inhibited Wnt signaling by targeting the transcription complex and was shown to interfere with both β-catenin/Tcf4 and β-catenin/LEF1 in dose-dependent manners in protein extracts from HCT116 cells. Compound 10 was examined for disruption of the interaction between β-catenin and E-cadherin. In fact, common armadillo repeats [10, with β-catenin immunoprecipitated from protein extracts of HCT116 cells. Compound 10 reduced cell motility, cell-cycle progression and the overexpression of many Wnt target genes . Compound 10 blocked the self-renewal capacity of cancer stem cells (CSCs), a significant result since high Wnt/β-catenin activity characterizes colon CSCs and other CSCs [10 reduced tumor growth and induced differentiation of colon CSCs in a mouse xenograft model. The authors hypothesized that 10, through the negatively charged sulfonamide group, forms binding interaction with Asp16 of TCF4, and thus to the Lys435 of the positively charged pocket of b-catenin. The hydrophobic cleft of the b-catenin formed by residues Cys466 and Pro463 might host the hydrophobic tail of 10.Fang, et al. , performi’s rule . SARs st repeats ,33 drive repeats . The preher CSCs . Compoun12, 13 and 14 weakened the interaction of β-catenin treated with inhibitor to the N-terminal domain of Tcf4 and significantly inhibited β-cat-Tcf interactions. Computational studies based on the β-cat crystal structure highlighted key contacts of β-catenin: Lys312 and Lys345 with Glu16 on Tcf; Lys435 with Asp16 on Tcf; and Phe253 and Phe293 with Leu48 on Tcf [12 was significantly superior to the previously reported inhibitor Calphostin C, a PKC inhibitor that also antagonizes the Tcf/β-catenin complex with a similar inhibitory mechanism. Compounds 12–14 showed potent inhibition in the luciferase-based Wnt reporter assay, disrupted the interaction of β-catenin with Tcf4 without affecting its interaction with E-cadherin and α-catenin, inhibited the transcription of the downstream target genes of β-catenin, such as WISP-1, Axin-2, CycD1, and c-Myc and a variety of Wnt-responsive phenotypes. iCRTs 12–14 had an efficacy comparable with fluorouracil (5-FU) in human CRC. Computational studies revealed that 12 and 13 form highly specific electrostatic contacts with Lys435 and Arg469 of TCF and β-catenin. The carboxamide NH of compound 12 forms a hydrogen bond with Arg469 on β-catenin, a residue that is critical for stabilizing β-cat-TCF4 interactions. The carboxylic group of 13 mimics interactions mediated by the side chain of Asp16 on TCF4 with Lys435 on β-catenin [The Gonsalves research group set an assay for HTS to identify small molecule candidates that would induce phenotype in the absence of dAxin as a consequence of the loss of β-catenin or Drosophila Tcf (dTcf) . The aut8 on Tcf ,38. Muta8 on Tcf . The oxa-catenin .15) [C-terminally biotinylated human Tcf4 (residues 7–51) and N-terminally His6-tagged human β-catenin (residues 138–686) fragments. In the FP assay, the authors used fluorescein-labeled human Tcf4 at the C-terminal (residues 7–51) to form a complex with human β-catenin (residues 138–686), emitting polarized light. Compound 15 inhibited the TOPFlash luciferase activity in pcDNA3.1-β-catenin-transfected HEK293 and SW480 cells with the IC50 values of 0.86 and 0.71 μM, respectively, and the growth of SW480, HCT116 and HT29 cells with low micromolar IC50 values. However, 15 showed off-target effects at high concentrations due to the presence of both the acyl hydrazone moiety, a PAINS substructure [15 was converted to an amide 16. In the AlphaScreen and FP assays, 16 was similar to that of 15, and V511, I569 and R469 were indicated as key residues by site-directed mutagenesis studies. Further optimization resulted in compound 17 yielding IC50 of 26 μM in the TOPFlash and FOPFlash luciferase reporter assay using pcDNA3.1-β-catenin-transfected HEK293 cells. Compound 17 blocked the transactivation of canonical Wnt signaling, suppressed the expression of Wnt-specific target genes and inhibited the growth of SW480 and HCY116 cancer cells with IC50 of 2.0 and 31 μM, respectively. The cell-based inhibitory activities correlated with the biochemical assays. The Kd value of the β-catenin/Tcf PPI suggested a significant selectivity over the β-catenin/cadherin and β-catenin/APC PPIs. Site-directed mutagenesis and SAR studies defined the binding mode of these inhibitors with β-catenin. These results showed that targeting the Tcf4 G13ANDE17 binding site of β-catenin with small molecules can lead to selective inhibition of the β-catenin/Tcf PPIs [Catrow’s group discovered six natural products of β-catenin/Tcf inhibitors though HTS of 52,000 compounds ,41. Opti15) , a new s15) . The Alptructure , and theTcf PPIs .18) was identified as a Tcf4 competitor. It showed KD of 450 nM in direct-binding ITC experiments and specific inhibition for Tcf-4 transactivation in the cellular luciferase reporter system. Binding modes were proposed for 18 and two analogs identified through a similarity search. Analog 1, with decreased affinity, lacked the methyl group at position 2 of the furane, and analog 2, with a piperidine moiety in place of the distal phenyl group that was not a competitor with Tcf 4. The proposed binding mode of 18 to the Lys435/Lys469 hot spot of β-catenin highlighted two pockets, with Asp16 of Tcf4 and with the groove of β-helices 23 and 30 of the armadillo repeat. One of the pockets contains the methylfurane where the methyl group laid in a narrow cleft, whereas the other pocket contains the phenyl moiety [Trosset, et al., performed docking studies of a 17,700-compound collection of Pharmacia at Nerviano Medical Sciences, Italy, using the QXP program . The aut19) exhibited a Ki of 3.14 μM as a disruptor of β-catenin/Tcf interactions. In the ITC assay, 19 showed Kd of 0.531 μM to wild-type β-catenin, and in the AlphaScreen assay, displayed a Ki of 7.60 μM. Both the FP and AlphaScreen assays indicated that 19 completely disrupted β-catenin/Tcf4 PPIs. The ITC indicated Lys435 of β-catenin is critical for binding to 18. The Kd values, Arg469Val of 3.21 μM (a mutation does not affect the binding of β-catenin to Tcf4) and Lys508Ala of 1.59 μM, showed that these residues involved in the binding of 18 to β-catenin  exhibited a Ki value of 1.32 ± 0.56 μM against the wild type β-catenin and completely disrupted the β-catenin/Tcf PPIs. The carboxylic acid 20 exhibited dual selectivity for β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC interactions. The corresponding ethyl ester UU-T03 (21) easily crossed the cell membrane and inhibited β-catenin/Tcf PPIs in a dose-dependent manner but had no effect on β-catenin/cadherin and β-catenin/APC PPIs. In the Wnt-responsive luciferase reporter assay, 21 yielded IC50 of 28.7 and 37.6 μM in SW480 and Wnt-activated HEK293 cells, and in the MTs assay on the growth of SW480, HCT116 and HEK293 colorectal cancer cells, it yielded IC50 values of 10.8, 11.0 and 84.0 μM. Site-directed mutagenesis studies to evaluate the binding mode of 20 with β-catenin highlighted the importance of the hydrophobicity of pocket B of the Tcf4 G13ANDE17 binding site for inhibition. The guanidino group of Arg474 was found to be more important than that of Arg515 for binding with 20. The deletion of two gaunidino groups of Arg474 and Arg515 remarkably reduced the β-catenin inhibitory activity of 20.Alanine scanning and biochemical assays led Huang and co-workers to identify a selective binding site that can differentiate β-catenin/Tcf, β-catenin/cadherin and β-catenin/APC interactions . Compari22) [22 inhibited β-catenin/Tcf4 luciferase activity and decreased mRNA expression of Cyclin D1 and Axin2, while it attenuated H-ras/β-catenin-induced foci formation. The direct binding of 22 with β-catenin disrupted the interaction between β-catenin and Tcf4 in vitro. Compound 22 decreased the growth of a patient-derived xenograft (PDX) colon cancer with a high expression level of β-catenin. The key role of the nitrogen atom at position 3 of the benzimidazole ring for the interaction of 22 with β-catenin was predicted by molecular modeling studies and confirmed in the DLD-1 cell proliferation and β-catenin/Tcf4 luciferase assays. The nitrogen at position 3 of the benzimidazole forms a hydrogen bond with β-catenin and its replacement with a carbon atom decreased the activity of 22 against DLD1 colon cancer cell growth.Shin’s group designed benzimidazole HI-B1 (22)  as a cyc22) . In DLD-50 < 10 μM: PKF115-584, CGP049090 and PKF222-815 were isolated from fungal organisms; PKF118-744 (23), PKF118-310 (24) and ZTM000990 (25) originated in Actinomycete strains; NPDDG39.024 was isolated from the marine sponge Fascaplysinopsis reticulata; and NPDDG1.024 was derived from NCI collections. In the ELISA, compounds 15–17 inhibited the Tcf4/β-catenin association by 50% with IC50 values of 2,4, 0.8 and 0.64 mM, respectively. The selected compounds showed antagonism to the β-catenin-dependent activities, including the expression of c-Myc or Cyclin D1, cell proliferation, and duplication of the Xenopus embryonic dorsal axis. The authors report that molecular mechanisms by which the isolated compounds interfere with the targeted interaction are unclear. In multiple myeloma (MM), PKF115-584 (26) blocked expression of Wnt target genes and induced cytotoxicity in both patient MM cells and MM cell lines without a significant effect in normal plasma cells. In xenograft models of human MM, 18 inhibited tumor growth and prolonged survival [Lepourcelet screened approximately 7000 purified natural compounds from proprietary and public collections to identify compounds that disrupt f/β-catenin complexes . The autsurvival .27) is an ent-kaurane diterpenoid isolated from Isodon rubescens var. lushanensis, a plant used in folk medicine. Li reported that 27 selectively inhibited the growth of human CRC cells with a GI50 value in the nanomolar range. The mechanism of action of 27 was to antagonize the Wnt-signaling pathway. Compound 27 reduced the expression of Cyclin D1 and c-Myc, and induced G1/S phase arrest in HCT116 cells by impairing the association of β-catenin/Tcf4 transcriptional complex, likely through directly blocking the binding of β-catenin to Tcf4 [27 did not affect the stabilization of β-catenin or its nuclear translocation. It strongly inhibited the binding of TCF4 to β-catenin in a dose-dependent manner in SW480 cells and in the in vitro binding assays with purified recombinant β-catenin and TCF4 proteins.Henryin ( to Tcf4 . Compoun28) was identified as a dose-dependent inhibitor of the β-catenin/Tcf4-driven luciferase activity in both primary HCT116 and pcDNA3.1-β-catenin-transfected HEK293 cell lines. Compound 28 decreased the HCT116 cell viability, at 5 μM, it inhibited > 80% colony forming activities and was not cytotoxic on normal HEK293 and HUVEC cells. Site A was sufficiently wide to adopt a small molecule that could disrupt β-catenin/Tcfs interactions. Lys435, Arg469, Lys508, Arg515 and Glu571 were key polar residues for binding of β-catenin with Tcf4. Important interactions on site A were Asp16 of Tcf4 with Lys435 and Asn430; Glu17 of Tcf4 with Lys508; and Ile19 and Phe21 of Tcf4 side chain with Arg386 aliphatic portion of β-catenin. Compound 28 was close to the Ile19 and Phe21 residues of Tcf4, and formed hydrophobic interactions with Pro463 ring of the β-catenin hydrophobic cleft. The other half of 28 laid close Asp16, also interfering with the β-catenin/Tcf4 interaction  and aStAx-35R  (30) inhibited competitively the binding of β-catenin to GST-TCF4(1-52) in the presence of a N-terminal acetylation. The crystal structure of N-terminally acetylated 29 in complex with residues 134–665 of β-catenin was resolved at 3.0 Å resolution. The β-catenin/29 stapled peptide crystal structure was deposited under PDB 4DJS. The binding site on β-catenin of 29 was revealed using the unliganded β-catenin structure PDB 1QZ7. The Trp residues at positions 468 and 481 increased the binding affinity for β-catenin engaging recognition pockets of the surface of β-catenin. Trp481 formed interactions with Trp338 of β-catenin and contributed to a H-bond with the carboxyl oxygen of Asp299 in β-catenin. Compound 30 proved to bind at the same Axin site but did not increase steady-state levels of β-catenin. Both 29 and 30 suppressed luciferase activity; the proliferation of DLD1 and SW480 cells was blocked by 30. The panel of canonical Wnt-driven genes was strongly suppressed, indicating that 30 antagonizes the nuclear form of β-catenin and hampers transcriptional coactivation for TCF proteins [StAx peptides were investigated for the ability to compete with TCF4 for binding of β-catenin by means of an immobilized GST-tagged CBD of TCF4(1-52). Stapled peptides aStAx-35 (m.w. 788.8) (proteins .31) from rosemary, that inhibited, in a dose-dependent manner, the binding of β-catenin to BCL9 in vitro, and β-catenin-dependent transcription in CRC cells [31. The mechanism of action of 31 was to promote selectively the proteasomal degradation of unphosphorylated β-catenin in an H1-dependent manner. To identify inhibitors of this interaction, the authors developed an in vitro assay that monitors the binding of His-HD2 (BCL9 homology domain 2) to glutathione S-transferase (GST)-ARD (immobilized on glutathione-coated microplates), using a colorimetric assay to quantify bound His-HD2 after addition of compounds. NMR saturation transfer difference (STD) spectroscopy was used to investigate the interaction of 31 with its target domain using the minimal HD2-binding domain within its N-terminus (four repeats: R4) [31 on R4 could explain its in vivo effects on β-catenin. The authors proposed that the metastable H1 also predisposed β-catenin to low-grade aggregation in vivo, and that this is exacerbated by 31, which could earmark β-catenin for proteasomal degradation. Saturation transfer difference (STD) NMR spectroscopy assays were performed to indentify the binding domain of 31 with purified R4 or BCL9 homology domain 2 (HD2). R4 tested positive in this ligand-observed binding assay whereas HD2 was negative. Titration with varying concentrations of R4 allowed researchers to estimate a Kd in the 5–20 μM range. The STD assays unequivocally identify R4 as the molecular target of 31.De La Roche and co-workers identified some natural compounds, including the carnosic acid (RC cells . Biophysats: R4) . Evidenc32 was provided with two positively-charged pyrrolidino groups and a substituted phenyl ring were introduced to improve the selectivity between α-helix-mediated PPIs. AlphaScreen and ITC studies consistently showed that 32 bound wild-type β-catenin but not BCL9. Compound 32 inhibited the β-catenin/BCL9 PPI, but not β-catenin/E-cadherin PPI, in colorectal SW480, HCT116, HT29 CRC cells and triple-negative breast cancer cells MDA-MB-231 and MDA-MB-436, all overexpressing the Wnt signaling with IC50 values in the low micromolar range. In the AutoDock predicted the binding conformation within β-catenin (PDB 2GL7); main contacts were the H-bonds of the protonated pyrrolidino groups with D145 and E155, the interactions of the mono and difluoro substituted phenyl rings with L178 and L159, respectively, and the contact of the oxygen atom linked to one pyrrolidino group with L156.Hoggard and co-workers designed and synthesized novel small-molecule inhibitors that selectively disrupted β-catenin/BCL9 over β-catenin/cadherin PPIs . The bin33) showed the most potent in vitro activity and exhibited stronger binding affinity to β-catenin compared to BCL9-HD2A. In the homogeneous time resolved fluorescence (HTRF) binding assay, hsBCL9CT-24 yielded a Kd value of 4.21 nM, compared to Kd value of 192.3 nM of SAH-BCL9B. In a modified AlphaScreen that allows greater surface area of β-catenin/BCL9 binding [33 into the β-catenin hydrophobic pocket (PDB 3SL9) performed by GlideXP Maestro Schrodinger highlighted binding interactions with Ala187, Asp145, Leu178, Met174, Lys170 and Val 167 amino acids in the β-catenin hydrophobic pocket  with promising inhibition of the Wnt pathway . To impr binding ,66 to dec pocket .1–33 are summarized in Great efforts are underway to discover direct inhibitors of β-catenin as anticancer agents. The direct inhibition of the oncogenic β-catenin represents an intriguing approach to ameliorate the efficacy and tolerability of the anticancer therapy, compared to upstream effectors of the Wnt/β-catenin signaling pathway. However, various difficulties hinder the progression to clinical studies of these agents, including the lack of co-crystal structure of small molecules in complex with β-catenin, the suboptimal metabolic and physiochemical profile of the known β-catenin inhibitors and the emergence of unwanted off-target effects. Continuous efforts to develop inhibitors of the Wnt/β-catenin signaling pathway highlight the potential of this strategy for the treatment of cancer. The drug discovery on novel β-catenin agents urgently needs information of binding sites, binding modes, and selectivity over other proteins. Beyond that, we should consider the complexity of the cancer development involving cross-talk between cellular signaling pathways, multiple factors and gene mutations. Advancement in structural information of β-catenin as well in knowledge about the Wnt pathway and the cross-talk mechanisms involved in normal and pathological conditions, we are confident, will pave the way for the development of effective β-catenin-targeting anticancer agents. Biological assays used to evaluate the β-catenin activity and activities of compounds


Spinal Muscular Atrophy (SMA) is a genetic neuromuscular disorder caused by reduction of the ubiquitously expressed protein Survival Motor Neuron (SMN). Low levels of SMN impact on spinal cord motoneurons (MNs) causing their degeneration and progressive muscle weakness and atrophy. To study the molecular mechanisms leading to cell loss in SMN-reduced MNs, we analyzed the NF-κB intracellular pathway in SMA models. NF-κB pathway activation is required for survival and regulates SMN levels in cultured MNs. Here we describe that NF-κB members, inhibitor of kappa B kinase beta (IKKβ), and RelA, were reduced in SMA mouse and human MNs. In addition, we observed that Gemin3 protein level was decreased in SMA MNs, but not in non-neuronal SMA cells. Gemin3 is a core member of the SMN complex responsible for small nuclear ribonucleoprotein biogenesis, and it regulates NF-κB activation through the mitogen-activated protein kinase TAK1. Our experiments showed that Gemin3 knockdown reduced SMN, IKKβ, and RelA protein levels, and caused significant neurite degeneration. Overexpression of SMN increased Gemin3 protein in SMA MNs, but did not prevent neurite degeneration in Gemin3 knockdown cells. These data indicated that Gemin3 reduction may contribute to cell degeneration in SMA MNs. Survival Motor Neuron 1 (SMN1) gene and a nearly identical gene, SMN2, is always present  is an autosomal recessive neuromuscular disease characterized by the loss of function of spinal cord motoneurons (MNs) leading to muscle weakness and atrophy . The dis present . SMN1 ansplicing . The tru2 copies . Inactivethality . Despiteethality .Reduction in SMN protein affects many cellular and molecular pathways, including the biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNP) . SMN is The protein component of gems number 3  is multifunctional, binding RNA secondary structures, regulating RNA metabolism, and performing other roles that do not directly involve RNA . Gemin3 In this study, we examined the NF-κB pathway and regulation of Gemin3 and SMN in several SMA models. Results clearly indicated that IKKβ, RelA, and Gemin3 were reduced in cultured SMA MNs and in SMA mouse postnatal spinal cord protein extracts. Nevertheless, when the level of these proteins was analyzed in non-neural SMA tissues, results showed differences from MNs observations. In SMA MNs, Gemin3, and SMN levels are interdependent: endogenous reduction of Gemin3 reduced SMN and vice versa. We also observed that endogenous reduction of Gemin3 induced neurite degeneration, which cannot be prevented by SMN over-expression. In summary, neurons and non-neuronal cells from SMA models displayed different molecular signs of intracellular deregulation and Gemin3 alterations observed in SMA MNs may contribute to understand some molecular mechanisms involved in SMA cell failure, such as neurite degeneration.89AhmbSmn1TM1Msd/J (mutSMA) (RRID:IMSR_JAX:005024) and FVB⋅Cg-Grm 7Tg(SMN2)89Ahmb Smn 1TM1Msd Tg(SMN2*delta7)4299Ahmb/J (SMNdelta7) (RRID:IMSR_JAX:005025). mutSMA  and SMNdelta7  were obtained by crossing heterozygous  animals. Littermates mutSMA/SMNdelta7 and control  were used for the experiments.Experiments involved two severe SMA mouse models, FVB⋅Cg-Tg (SMN2)For MN purification or postnatal experiments, a fragment of the head or tail was obtained for genotyping. The REDExtract-N-Amp Tissue PCR Kit  was used for genomic DNA extraction and PCR setup, with the following primers: WT forward 5′-CTCCGGGATATTGGGATTG-3′, SMA reverse 5′-GGTAACGCCAGGGTTTTCC-3′, and WT reverse 5′-TTTCTTCTGGCTGTGCCTTT-3′. Birth was defined as postnatal day 0 (P0).All procedures were done in accordance with the Spanish Council on Animal Care guidelines and approved by the University of Lleida Advisory Committee on Animal Services (CEEA02-01/17).Motoneurons primary cultures were obtained from the spinal cords of CD1 or SMA mouse embryos at E13 essentially as described . IsolateMorphometric analysis of neurite degeneration was performed as described  with modHuman fibroblast cell lines were obtained from the Coriell Institute for Medical Research . The Coriell Cell Repository maintains the consent and privacy of the donor samples. All cell lines and culture protocols in the present study were carried out under institutional review board guidelines at University of Lleida and the IRBLleida research center.2 in 35 mm tissue-culture dishes and maintained in supplemented MEM. Two days later, total cell lysates were collected and submitted to western blot analysis.Two human fibroblast cell lines from patients with SMA  and one unaffected control subject  were purchased and cultured following manufacturer instructions. Cells were maintained in Eagle’s Minimum Essential Medium (EMEM) (Sigma) supplemented with non-inactivated fetal bovine serum  (15% v/v), 0.5 M of L-Glutamine (Gibco), non-essential amino acids (Gibco) (1% v/v), and 20 μg/ml Penicillin-Streptomycin (Gibco). Cells were subcultured every 3–4 days. For western blot analysis, cells were plated at 3,000–4,000 cells/cm2 laminin-coated glass coverslips for immunofluorescence experiments .The human iPSCs used in the present work were purchased from Coriell Institute for Medical Research or Cedars Sinai Biomanufacturing Center. The GM23411*B (Coriell) iPSC cell line  was considered the control condition, the GM23240*B (Coriell) iPSC cell line from a patient with SMA type 2  was considered the SMAII condition, and CS84iSMA-nxx  iPSC cell line from a patient with SMA type 1  was considered the SMAI condition. Control and SMA cells were differentiated to MNs as described  with min°C. Samples were disaggregated using Direct Quant 100ST Buffer  and a G50 Tissue Grinder . Total cell lysates of cultured cells or tissue homogenates were resolved in SDS polyacrylamide gels and transferred onto polyvinylidene difluoride Immobilon-P transfer membrane filters , using an Amersham Biosciences  Semi-dry Trans-blot. The membranes were blotted with anti-SMN ; anti-Gemin3 (12H12)  and anti-Gemin3 (D-5)  for human and mouse samples, respectively, both from Santa Cruz Biotechnology, Dallas, TX, USA; and anti-IKKβ (D30C6) , and anti-NF-κB p65 (L8F6) (RelA) , both from Cell Signaling Technology, Danvers, MA, USA. To control the specific protein content per lane, membranes were reprobed with monoclonal anti-α-tubulin antibody . Blots were developed using LuminataTM ForteWestern HRP Substrate (Millipore).Western blots were performed as previously described . For pos2 Fragment Donkey anti-Sheep IgG (H + L), 1:400, Cat. No. 713-166-147 from Jackson ImmunoResearch, West Grove, PA, USA. Hoechst  staining was performed to identify nuclear localization in cell soma. Samples were mounted using Mowiol  medium. Microscopy observations were performed in a FV10i Olympus confocal microscope . Quantification of fluorescence intensity was performed blinded, using the NIH ImageJ software  for 10 min and with cold methanol (Sigma) for 30 additional sec. Cells were permeabilized with 0.2% Triton X-100 and incubated for 2 h with 5% bovine serum albumin (BSA) in PBS. Primary antibody  was diluted in 0.2% Triton-X-100 and incubated overnight with 5% BSA in PBS. After washing, the secondary antibody was added: anti-mouse ALEXA555, 1:400, Cat. No. A21422 and anti-rabbit ALEXA488, 1:400, Cat. No. A11008, both from Invitrogen, Waltham, MA, USA and Cy™3 AffiniPure F(ab’)software . CorrectSMN interference, constructs were generated in pSUPER.retro.puro  using oligonucleotides that target the SMN sequence, indicated by capital letters as follows: shSMN, forward: gatccccCGACCTGTGAAGTAGCTAAttcaagagaTTAGCTACT TCACAGGTCGttttt, and reverse, agctaaaaaCGACCTG TGAAGTAGCTAAtctcttgaaTTAGCTACTTCACAGGTCGggg. Constructs were subcloned in pLVTHM vector containing the Green Fluorescence Protein (GFP). The shRNA lentiviral plasmids (pLKO.1-puro) for human/mouse Gemin3 were purchased from Sigma. The RefSeq used were NM_017397, corresponding to mouse Gemin3, and NM_007204, corresponding to human Gemin3. The clones TRCN0000009650 and TRCN0000007888 were designed for mouse and human interference of Gemin3, respectively.Lentiviral-based vectors for RNA interference-mediated gene silencing were performed as described . For SMNLentiviral particles were propagated in human embryonic kidney 293T (HEK293T) cells using the polyethylenimine  cell transfection method. Plasmids containing the shRNA sequence or the empty vector, pSPAX2 and pM2G were cotransfected to HEK293T; lentivirus titer and MN transduction were performed as described previously .SMN1 cDNA (NCBI accession number NM000344) was amplified by PCR from pDNR-LIB vector (Invitrogen) using the following primers, forward: cacaggatccatggcgatgagcagcggcggc; and reverse: tgtgggatccttaatttaaggaatgtgagca (underline indicates BamH1 restriction site)  to monitor transduction efficiency. Lentivirus containing over-expression constructs was obtained as described above.For SMN over-expression, the open reading frame of the human on site) . The PCR® Mini Kit  according to manufacturer instructions. Eighty nanograms of total RNA from each condition were used for each individual qRT-PCR reaction. The assays were performed in a CFX96 Real-Time System  using iTaq™ Universal SYBR® Green One-Step Kit from Bio-Rad. Real-Time was performed using human RelA specific primers: Forward (5′-CGAGCTCAGTGAGCCCATG-3′) and Reverse (5′-GGCACAGCAATACGCCGGG-3′) or Gemin3 specific primers: Forward (5′-GCTGGCCGTTTTGGTACATTG-3′) and Reverse (5′-TGCACAGCAGCTTTAACTTCC-3′), and specific primers of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward (5′-TGCACCACCAACTGCTTAG-3′) and reverse (5′-AGAGGCAGGGATGATGTTG-3′) as internal control. Quantification was completed using Bio-Rad CFX Manager real-time detection system software . Each sample was measured in triplicate; relative fold change gene expression levels were calculated with the formula 2^–(ΔΔCq).For qRT-PCR experiments, control and SMAII iPSCs were differentiated to MNs. Neurospheres were dissociated and plated in laminin-coated six-well tissue-culture dishes at a density of 400,000 cells/well in MN maturation medium. After 6 days, total RNA was extracted using the RNeasyt-test or one-way ANOVA with Tukey’s multiple comparisons or Dunnett’s multiple comparisons test for all other analysis. Similar variances between the compared groups were assumed. Values were considered significant when p < 0.05.All experiments were performed at least three independent times. Values were expressed as mean ± estimated standard error of the mean (SEM). Statistical analysis was done with GraphPad Prism, version 8 (graphPad Software Inc.). Differences between groups were assessed by two-tailed Student p = 0.0049) and in P5 SMNdelta7  samples, compared to their respective controls  compared to the non-affected control  and P5 SMNdelta7  extracts, compared to the corresponding controls , compared to the clinical unaffected control  and quadriceps (SMNdelta7) were dissected from control and mutant genotyped mice at postnatal stage P2 (mutSMA) and P5 (SMNdelta7) and protein extracts were submitted to western blot analysis. IKKβ protein level was significantly reduced in P2 mutSMA  and SMNdelta7  protein extracts compared to their respective controls (p < 0.0001) and SMAI  differentiated MNs compared to the non-affected control (RelA gene expression we quantified RelA messenger RNA (mRNA) by quantitative RT-PCR (qRT-PCR). Gapdh gene was used as a control. SMAII and control iPSCs were differentiated to MNs. Total RNA from 7 days differentiated MNs was extracted and reverse-transcribed to cDNA, used as a template to quantify RelA transcript level. Results indicated that RelA mRNA expression  was increased in SMAII cells compared to control , compared to the non-deprived control nuclei (76.39 ± 4.68). When 24 h deprived cells were stimulated in the presence or the absence of neurotrophic factors, fluorescence measures indicated that IGF and CNTF treatment induced nuclear relocation of RelA   . Togethep = 0.8101; quadriceps 0.911 ± 0.14, p = 0.5321) or SMA human fibroblasts , compared to their respective controls.We next analyzed RelA in SMA cultured human fibroblasts, P2 mouse mutSMA gastrocnemius, and P5 mouse SMNdelta7 quadriceps. As shown in p < 0.0001) and SMAI  MNs, compared to the control condition  compared to control , compared to control condition  , compared to control condition , in mouse SMNdelta7 quadriceps , or in gastrocnemius mutSMA , compared to their respective controls. These results together revealed divergences in Gemin3 protein expression in SMA cells and tissues.To study whether Gemin3 deregulation in SMA models is present in neurons and non-neuronal cells, we analyzed Gemin3 protein level by western blot and immunofluorescence using an anti-Gemin3 antibody. SMA and control human iPSC cells were differentiated to MNs and 7 days protein extracts were obtained. Gemin3 protein level was significantly reduced in human SMAII  . Togetheroblasts . Resultsondition . Neverthp < 0.0001; Gemin3 0.684 ± 0.078, p = 0.0038, respectively). Results indicated that shSmn treatment significantly reduced Gemin3 level  (p < 0.0001) and IKKβ  protein level, compared to their respective EV controls  . Furthercontrols .p < 0.0001; SMAII: 0.66 ± 0.089, p = 0.0051), compared to the EV transduced controls (p = 0.0002) and SMAII  conditions  and SMAII  cells, compared to their respective EV controls. This suggests a direct association between Gemin3 and SMN, IKKβ, and RelA level in cultured human MNs.MN progenitors (MNPs) cells derived from human non-affected control and SMAII iPSCs were plated in MN differentiation medium and transduced with lentiviral particles containing shGemin3  or ovSMNcontrols . SMN pronditions . When NFnditions  and RelAnditions  protein p = 0.0031) and SMAII  cells compared to the EV condition, indicating that overexpression was accomplished. When Gemin3 and IKKβ were analyzed in ovSMN treated cultures, the protein level was significantly increased in control  and SMAII  cells, compared to their EV respective controls , indicating that SMA MNs displayed neurite degeneration in vitro. Nevertheless, when SMN protein was increased (SMNdelta7 ovSMN condition) we observed a reduction in the percentage of neurite degeneration (41.56 ± 4.117) compared to non-transduced (p = 0.0051) or EV  controls, suggesting that neurite degeneration in SMA cells was the consequence of SMN reduced protein levels. Interestingly, endogenous knockdown of Gemin3 protein caused a significant increase of neurite degeneration in control  and SMNdelta7 , compared to their respective EV controls. Nevertheless, overexpression of SMN did not prevent neurite degeneration caused by Gemin3 reduction . The percentage of degenerating neurites (swelling and blebbing) was considered with respect to the total number of neurites in the microscope field. To determine the presence of neurite degeneration in SMNdelta7 MNs, cells were isolated from 12.5 days embryos and transduced with lentivirus carrying shGemin3 or ovSMN or shGemin3 plus ovSMN or EV or left untreated. After 12 days in culture, SMNdelta7 non-transduced cultures showed an increase of neurite degeneration compared to the control non-transduced condition -mediated activation of IKKβ . Gemin3 C. elegans model pharyngeal pumping rate defects were not ameliorated after mel-46 (C. elegans Gemin3 ortholog) increase, nevertheless, neuromuscular signaling was improved by partially restoring levels and localization of synaptic proteins -linked proteins, reinforcing the link between ALS and SMA pathologies. Disruption of either TDP-43 or FUS enhances defects associated with Gemin3 loss-of-function, and Sod1 depletion is responsible of motor decline in Gemin3 mutant ult life . Both SMult life . An inveult life . Severalult life . Additioult life . Moreovee of SMN . In our RelA, Gemin3, and IKKβ mRNA in the somatodendritic compartment . However, these changes were not apparent in SMA fibroblasts or muscle, the non-neural tissues analyzed. Gemin3, a member of the SMN complex, was also reduced in SMA isolated MNs and spinal cord. Within this framework, an important aspect to consider is that Gemin3 reduction causes neurite degeneration that cannot be rescued by SMN increase. In addition, Gemin3 reduction in SMA cells may contribute to degeneration through NF-κB inhibition, exacerbating SMA phenotype. Gemin3 may act as a modifying factor for SMA disease and studies of Gemin3 restoration in SMN-reduced MNs will contribute to understand its therapeutic significance.In summary, the present study describes decreased levels of two members of the NF-κB survival pathway, IKKβ and RelA, in SMA isolated MNs The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by University of Lleida Advisory Committee on Animal Services (CEEA02-01/17).MPM: conceptualization, investigation, formal analysis, writing—original draft, and visualization. AS and MB: investigation, formal Analysis, and validation. AG and RS: conceptualization, supervision, writing—original draft, reviewing, visualization, and funding acquisition. All authors contributed to the article and approved the submitted version.


Although major advances were achieved in colorectal cancer (CRC) therapy, major concerns still remain on proper control of cancer metastasis and chemo-resistance in order to achieve satisfactory general treatment response. Previous studies suggested that OTUB1  serves as regulator of gene ubiquitination and participates in the pathogenesis of multiple malignancies. Therefore, to discover its molecular mechanism in CRC tumor growth and metastasis will contribute in CRC treatment strategy development. Clinical tissues and CRC cancer cell lines were utilized to evaluate OTUB1 expression pattern. Functional tests including cellular proliferation, migration and invasion, as well as chemo-resistance, etc., were evaluated to investigate the role of OTUB1/β-catenin regulatory pathway on CRC malignant biological behaviors. Both CRC tumor tissues and CRC cell lines exhibited promoted OTUB1 expression level. Subsequent experiments further suggested that OTUB1 promoted CRC malignancy by enhancing protein stability of β-catenin, via inhibition of its protein degradation by UPP pathway, which indicated its crucial role in enhancement of CRC tumor cellular proliferative and chemo-resistant capabilities. This study reported that OTUB1 exhibited novel pro-survival and pro-metastatic function by interaction of β-Catenin via Ubiquitin-proteasome pathway. Our research indicated that OTUB1/β-Catenin regulatory axis might be potential druggable target for CRC cancer patients’ treatment.  Despite the vast advances that have been made in the era of oncological immunotherapy, surgical operation, combined with chemotherapy, are still applied as the foundation of CRC treatment . AlthougMultiple researches have demonstrated various dysfunctional signaling pathways, which play important roles in tumor chemo-resistance and distal organ invasion. Dysregulation of Wnt/β-Catenin signaling was proved to be involved in uncontrolled tumor expansion and differentiation inhibition . It was OTUB1  was also known as a K48 linkage-specific deubiquitinating enzyme that contains two distinct ubiquitin-binding sites . And K63In the present study, we suspected that OTUB1 might regulate CRC through targeting β-Catenin. We aimed to find the potential regulatory mechanism for how OTUB1 affect CRC via β-Catenin. We further delineated the OTUB1ʹs impact on Wnt/β-Catenin pathway, which might provide novel insight for future CRC treatment.2.2.1.CRC cancer patients diagnosed between 5/1/2020 to 11/1/2021 were retrospectively recruited in this study. Before sample collection, patients did not receive any prior surgery, radiotherapy or chemo-therapeutic agents. Post-surgical CRC tumor tissues and matched normal sample tissues were correspondingly gathered. Harvested samples were saved in lab for subsequent experiments. The study has been approved by the Ethics Committee of Fujian Medical University Union Hospital . Declaration of Helsinki was compliant in this research. Informed consents from the patients enrolled were obtained.2.2.RNA was extracted from cells and tissues by TRIzol agent . RT-PCR kit  was used according to the protocol for reverse-transcription. qRT-PCR was conducted utilizing primers listed in supplementary Table 1. Quantification results were further normalized to the level of GAPDH2.3.2.SW620, HCT116, SW480, RKO, LOVO, DLD1, FHC cells were saved in our research center. Cells were cultured in DMEM medium containing 10% fetal bovine serum in an incubator with 5% CO2.4.Western blot procedure was performed as described previously. The proteins were extracted using 1% PMSF and RIPA lysine buffers. SDS-polyacrylamide gels were used for lysates separation. Immunoblot assay was conducted with primary antibody against β-actin, β-catenin, CD44, CD133, OTUB1  overnight at 4°C. After washing with PBS, the proteins were incubated with second antibodies for 2 h at room temperature. ECL chemiluminescence kit  was used to detect protein. The protein gray was analyzed using Image J.2.5.For the cellular proliferation experiment, 2000 cells per group were seeded. After vector transfection, absorbance at 490 nm was evaluated. For subsequent assay, 3000 cells per group were seeded and transfected. Then, colonies were measured and repeated three times for subsequent quantification.2.6.For Transwell study, the procedure was according to the description previously reported elsewhere. As for wound healing experiment, each group of cells were placed and monolayers were scraped. The distance between wound was tested three times. The distance between the wound was repeatedly measured after incubation for 48 h.2.7.After treatment, cells were washed by PBS three times, and then cell samples were lysed in IP buffer. Afterward, lysate samples were then undergone centrifuge treatment and pre-clearing. Supernatants treated by 5 μg anti-FLAG) and A/G-Sepharose bead were further incubated at 4°C environment overnight. After that, lysis buffer was utilized for beads washing for three times and sodium dodecyl sulfate (SDS) loading buffer was used for resuspension. Western blot was subsequently performed using corresponding antibody2.8.First, cell samples were treated by PBS for washing, and were then treated with paraformaldehyde to fix. Afterward, after permeabilization, primary antibody was used for incubation. Subsequently, cells were incubated with conjugated secondary antibody. Then, the cells samples were stained by DAPI and examined by confocal microscopy for image generation2.9.Cell samples were treated by MG132  after 5 h of transfection. Then, cellular lysate was treated by lysis buffer and after treatment, each cell group proteins were immune-precipitated (IP) with corresponding antibodies. Each group of cell proteins were determined and measured by Western blot.2.10.2 and 37°C for 48 h. Cell transfection was conducted using Lipofectamine 2000 .The target genes were amplified via PCR. The target genes were inserted into pcDNA3.1 vector. The cells were planted into a 60-mm dish. After culture in the incubator with 5% CO2.11.CHX  was used in this study to perform CHX-chase test. CHX (14 μg/mL) was mixed with cells for 0, 2, 4, 8, respectively. Then, the protein was extracted, and protein expression levels were measured through western blotting.2. 12.Statistical analysis was performed with SPSS (22.0) software. Data were harvest from three independent experiments. P < 0.05 was considered as statistical significance. T-test and ANOVA test were used for p value determination3.3.1.In our research, we investigated OTUB1 expression utilizing immunohistochemical (IHC) test on tumor and matched adjacent tissue of CRC patients. As shown in 3.2.We aimed to detect the impact of OTUB1 modulation on CRC cancer cells. OTUB1-specific overexpression vectors and shRNA were designed and validated via qRT-PCR and WB experiments . Then, C3.3.In addition, in order to further explore the impact of OTUB1 expression modulation on CRC cancer cell migrative capabilities, wound healing test on DLD1 and HCT116 cell lines were performed. OTUB1 overexpression and OTUB1-specific shRNAs were applied in each cell group. As depicted in 3.4.Next, we conducted cellular viability test on HCT116 and DLD1 cells with OTUB1 gene modulations. Three different chemo-agents  with multiple concentrations were utilized. As a result, OTUB1 silencing significantly suppressed HCT116 cellular survivability, while OTUB1 overexpression significantly enhanced cancer cell viabilities . Moreove3.5.To clarify the target of OTUB1, we focused on the potential impact of OTUB1 on key regulatory gene in CRC pathogenesis, i.e. β-Catenin. We first retrospectively evaluated the protein and mRNA expression of β-Catenin and OTUB1 in our clinical cohort, and results indicated that OTUB1 protein expression was significantly associated with protein level of β-Catenin elevated the , while nNext, we applied CHX chase to detect protein degradation of OTUB1 and β-Catenin in HCT116 cell line. As shown in In the following research, we further validated the above-mentioned findings by designing β-Catenin specific overexpression vector . Through4.In this research, we discovered that OTUB1 upregulation was typical in CRC cancer cells. And OTUB1 exhibited multi-facet promotive effects on malignant biological behaviors of CRC cancer cell including tumor cell stemness, invasion and chemo-resistance. Meanwhile, OTUB1 as de-ubiquitinase was up-regulated in several human malignancies and OTUB1 participated in tumor pathogenesis via several different routes ,12. For β-Catenin has been extensively investigated as fundamental member of Wnt/β-Catenin canonical pathway. β-Catenin participates in several vital physiological processes ranging from embryo development to adult homeostasis. Cytoplasmic β-catenin is modulated by the destruction complex containing Adenomatous polyposis coli (APC) for final degradation ,17. Multin vitro and in vivo models with OTUB1 knock-out phenotype are of great value for further exploration.However, it is notable that several limitations exist in our research. As our clinical observation was based on limited-scale retrospective clinical samples, future studies with more-expanded cohorts are warranted to consolidate our findings in CRC patients. Besides, we generally detected the interaction between OTUB1 and β-catenin by cell line models. Future researches based on 5.In this article, we proved that OTUB1 expression up-regulation was characteristic in tumor cells of CRC cancer patients. OTUB1 exacerbated CRC malignancy by promoting the protein stability β-Catenin through inhibition of UPP pathway induced β-Catenin protein degradation. Our study offers novel clues of OTUB1ʹs role in CRC oncogenesis, and enlightens future study on targeted therapy development for CRC patients.Click here for additional data file.