Cellular and Molecular Life Sciences

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RICS and TICS evidence two diffusive subpopulations of single GlyR pentamers. A Representative confocal microscopy image of the first frame from an image series of a HEK293 cell expressing GlyR-α3L-eGFP. Frame-based intensity thresholding was used to remove GlyR clusters and the extracellular region from the analysis. Scale bar 10 µm. B Photon count values fluctuating along the yellow arrow in (A). C 3D autocorrelation with the gray outlining showing the average (ξ, 0) and (0, ψ) autocorrelation function. D) Average (ξ, 0) and (0, ψ) autocorrelation function and fit. Top graph displays the weighted residuals for the fit in the bottom graph. E Average diffusion constant and standard deviation obtained via RICS for GlyR-α3L-eGFP and GlyR-α3K-eGFP. F Representative TIRF microscopy image of the first frame from an image series of a HEK293 cell expressing GlyR-α3L-eGFP. Frame-based intensity thresholding was applied to remove GlyR clusters (indicated in red) and the extracellular region (indicated in light gray). Scale bar 10 µm. G Camera count
Co-localization and co-diffusion of GlyR-α3 isoforms in HEK293 cells at single-molecule expression confirms the presence of GlyR-α3L/K heteropentamers. A Representative dual-color TIRF image of a HEK293 cell co-expressing GlyR-α3L-eGFP (green) and GlyR-α3K-mCherry (red). Using intensity thresholding over the average of the 5 first frames the cell membrane was selected and bright regions containing GlyR clusters were omitted. Scale bar 10 µm. B Pearson's correlation coefficient of cells expressing GlyR-α3L-eGFP and GlyR-α3K-mCherry and cells co-expressing GlyR-α3L-eGFP and GlyR-α3L-mCherry plasmids. Both experimental groups have significantly higher co-localization compared to the negative control with GlyR-α3L-eGFP and Lyn-mCherry. Lyn is a monomeric membrane protein that does not interact with GlyR. The ρ values are shown for cells including (gray) and excluding (dark gray) GlyR clusters. C Spatial ρ from representative cells expressing GlyR-α3L-eGFP and GlyR-α3K-mCherry (left), GlyR-α3L-eGFP and GlyR-α3L-mCherry (middle) or GlyR-α3L-eGFP and Lyn-mCherry (right). D Representative dual-color TIRF image of a HEK293 cell co-expressing GlyR-
Single-channel measurements show activated α3K yields higher currents. A Transfected cells are identified by eGFP or mCherry fluorescence. Scale bar is 5 µm. B Left: Illustration of cell-attached single-channel electrophysiology: single receptors are exposed to glycine (30-80 µM) present in the patch pipette. Middle: example of a current time trace during which similar peak currents are seen. Right: A histogram of all measured currents is fit with a double Gaussian, yielding a best fit value for the mean baseline current and the mean peak current. Subtracting the mean baseline current from the mean peak current yields the mean current for the time
  • Veerle LemmensVeerle Lemmens
  • Bart TheveleinBart Thevelein
  • Yana VellaYana Vella
  • [...]
  • Jelle HendrixJelle Hendrix
Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson’s and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via cell-attached single-channel electrophysiology we show that heteropentamers exhibit currents in between those of K and L variants. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR-α3 channels, which is important to understand and tackle α3 related diseases.
 
Chemical structure of RES
The potential of RES in regulating the proliferation rate of BC cells. Different intracellular factors are affected by RES that led to inhibit the cancer cells proliferation and and initiate/promote cell death via activating apoptosis and autophagy mechanisms. It could induce apoptosis in cancer cells via improving three main mechanisms: increasing the intracellular oxidative stress, enhancing the expression level of p53, and by regulating mechanisms involved in caspase activation. On the other hand, it prevents autophagy via activating the mTOR pathway and preventing compounds related to the activation of autophagosome creation
Antimetastatic effects of RES against breast cancer cells. RES could exhibit its antimetastatic effects via suppressing both epithelial-to-mesenchymal transition (EMT) and DNA replication through the activation and suppression of different intracellular proteins and pathways. It could suppress molecules and pathways related to improving the EMT and DNA replication from one side and activate the inhibitors of EMT from the other side, that finally leads to the inhibition of cancer cell metastasis and reduce the invasiveness features of the cancer cells
RES loaded nanomaterials for BC treatment. RES could be encapsulated in or loaded on the surface of different types of nanomaterials. This could improve the therapeutic effects of RES via improving its bioavailability, delivering it to its targeted site, and releasing it in a controlled manner
  • Mitra BehroozaghdamMitra Behroozaghdam
  • Maryam DehghaniMaryam Dehghani
  • Amirhossein ZabolianAmirhossein Zabolian
  • [...]
  • Anupam BishayeeAnupam Bishayee
Breast cancer (BC) is one of the most common cancers in females and is responsible for the highest cancer-related deaths following lung cancer. The complex tumor microenvironment and the aggressive behavior, heterogenous nature, high proliferation rate, and ability to resist treatment are the most well-known features of BC. Accordingly, it is critical to find an effective therapeutic agent to overcome these deleterious features of BC. Resveratrol (RES) is a polyphenol and can be found in common foods, such as pistachios, peanuts, bilberries, blueberries, and grapes. It has been used as a therapeutic agent for various diseases, such as diabetes, cardiovascular diseases, inflammation, and cancer. The anticancer mechanisms of RES in regard to breast cancer include the inhibition of cell proliferation, and reduction of cell viability, invasion, and metastasis. In addition, the synergistic effects of RES in combination with other chemotherapeutic agents, such as docetaxel, paclitaxel, cisplatin, and/or doxorubicin may contribute to enhancing the anticancer properties of RES on BC cells. Although, it demonstrates promising therapeutic features, the low water solubility of RES limits its use, suggesting the use of delivery systems to improve its bioavailability. Several types of nano drug delivery systems have therefore been introduced as good candidates for RES delivery. Due to RES’s promising potential as a chemopreventive and chemotherapeutic agent for BC, this review aims to explore the anticancer mechanisms of RES using the most up to date research and addresses the effects of using nanomaterials as delivery systems to improve the anticancer properties of RES. Graphical abstract
 
  • Ying WangYing Wang
  • Jiang-Wei ZhangJiang-Wei Zhang
  • Jing-Wen WangJing-Wen Wang
  • [...]
  • Xiao-Ming DingXiao-Ming Ding
Early apoptosis of grafted islets is one of the main factors affecting the efficacy of islet transplantation. The combined transplantation of islet cells and bone marrow mesenchymal stem cells (BMSCs) can significantly improve the survival rate of grafted islets. Transcription factor insulin gene enhancer binding protein 1 (ISL1) is shown to promote the angiogenesis of grafted islets and the paracrine function of mesenchymal stem cells during the co-transplantation, yet the regulatory mechanism remains unclear. By using ISL1-overexpressing BMSCs and the subtherapeutic doses of islets for co-transplantation, we managed to reduce the apoptosis and improve the survival rate of the grafts. Our metabolomics and proteomics data suggested that ISL1 upregulates aniline (ANLN) and Inhibin beta A chain (INHBA), and stimulated the release of caffeine in the BMSCs. We then demonstrated that the upregulation of ANLN and INHBA was achieved by the binding of ISL1 to the promoter regions of the two genes. In addition, ISL1 could also promote BMSCs to release exosomes with high expression of ANLN, secrete INHBA and caffeine, and reduce streptozocin (STZ)-induced islets apoptosis. Thus, our study provides mechanical insight into the islet/BMSCs co-transplantation and paves the foundation for using conditioned medium to mimic the ISL1-overexpressing BMSCs co-transplantation. Graphical abstract
 
Contact-based pericellular interactions play important roles in cancer progression via juxtacrine signaling pathways. The present study revealed that hypoxia-inducible factor-1α (HIF-1α), induced even in non-hypoxic conditions by cell-to-cell contact, was a critical cue responsible for the malignant characteristics of glioblastoma multiforme (GBM) cells through Notch1 signaling. Densely cultured GBM cells showed enhanced viability and resistance to temozolomide (TMZ) compared to GBM cells at a low density. Ablating Notch1 signaling by a γ-secretase inhibitor or siRNA transfection resensitized resistant GBM cells to TMZ treatment and decreased their viability under dense culture conditions. The expression of HIF-1α was significantly elevated in highly dense GBM cells even under non-hypoxic conditions. Atypical HIF-1α expression was associated with the Notch1 signaling pathway in both GBM and glioblastoma stem cells (GSC). Proteasomal degradation of HIF-1α was prevented by binding with Notch1 intracellular domain (NICD), which translocated to the nuclei of GBM cells. Silencing Notch1 signaling using a doxycycline-inducible Notch1 RNA-interfering system or treatment with chetomin, a HIF pathway inhibitor, retarded tumor development with a significant anti-cancer effect in a murine U251-xenograft model. Using GBM patient tissue microarray analysis, a significant increase in HIF-1α expression was identified in the group with Notch1 expression compared to the group without Notch1 expression among those with positive HIF-1α expression. Collectively, these findings highlight the critical role of cell-to-cell contact-dependent signaling in GBM progression. They provide a rationale for targeting HIF-1α signaling even in a non-hypoxic microenvironment.
 
Osteogenic differentiation analysis by gene expression and extracellular matrix protein immunostaining. A Blue bars represent gene expression of Alpl, Col3a1, Bmp2 and Ibsp in GC condition, while red bars refer to RPM condition. Fold changes were calculated from the threshold cycles and expressed as the mean ± standard deviation. Student’s T test was applied for comparison against the gravity control GC. B Immunostaining at 28 days of differentiation showing collagen type I (COL1A1) marked in green with nuclei marked in blue on the left side of the panel and osteocalcin (BGLAP) marked in green with nuclei marked in blue on the right side of the panel, both in GC and RPM conditions. Blue bars represent the total fluorescence intensity (upper bars) and fluorescent area (lower bars) of the corresponding protein in GC. Red bars show the same parameters in the RPM condition. Statistically significant differences were assessed via Student T test (T tests: * = p < 0.05; ** = p < 0.01; *** = p < 0.001)
Mineralization assessment of BMSCs during osteogenic differentiation in GC and RPM. A ALPL enzymatic activity assessment at 4 time points: 8, 14, 21 and 28 days, in GC and RPM. B Optical microscope visualization of differentiating cells throughout the 28 days of experiment and crystal formation from the 14th day (inserts). C Maximum, minimum, and average crystal size values at 28 days are reported in the box plot and supplied with qualitative figures of the corresponding crystals. Statistics was calculated on more than 60 crystals per experimental point in images acquired with 20 × magnification in phase contrast via Student T test, comparing crystals in GC to RPM. D Frequency distribution of crystal size in GC and RPM conditions at 28 days. E Qualitative and quantitative representation of the alizarin red staining performed at 8 (T8) and 28 days (T28) in GC (blue histograms) and RPM conditions (red histograms). Significant differences were assessed via Student T test (T tests: * = p < 0.05; *** = p < 0.001)
Cytoskeletal reorganization investigated by immunostaining and bio-imaging analysis. A β-tubulin (green), F-actin (red) and nuclei (blue) were stained and visualized following 1 h, 1, 4, 8, 14, and 28 days of differentiation, both in GC and RPM conditions. B Graphs of F-Actin and β-tubulin mean intensity (T tests: * = p < 0.05). C Gray-scale images (1 and 2), fire lookup table (3 and 4) and concentric belts into cells (5 and 6) were used for the overall distribution of tubulin fluorescence intensity. The concentric distribution algorithm masks were used to quantify microtubules distribution per belt (5 and 6) and 15 cells per field were analyzed in three experimental replicates. D Graph of mean intensity and percentage of β-tubulin into each belt (Belt 0 = close to the centrosome; belt 10 = cell periphery) (T tests: * = p < 0.05)
Proteomics investigation. A Schematic representation of the experimental proteomics workflow, with indications of the software utilized in the different steps. B PCA of the sample replicates based on the protein groups intensities characterizing each sample. PC1 explains 35% of the variance and PC2 24%. C Hierarchical clustering of the top ten most downregulated and ten upregulated PGs D Pie chart showing DAPGs divided by their trend in RPM with respect to GC condition. E Following enrichment analysis of the DAPGs, a total of 90 pathways were found upregulated and 106 downregulated
Differentially abundant protein groups (DAGPs) categorization under major GO biological processes. The enriched GO_BPs were categorized under 10 major classes mainly involved in cell metabolism and cell fate. Data mapped are the Z-scored of the ratioed intensities
Microgravity-induced bone loss is a major concern for space travelers. Ground-based microgravity simulators are crucial to study the effect of microgravity exposure on biological systems and to address the limitations posed by restricted access to real space. In this work, for the first time, we adopt a multidisciplinary approach to characterize the morphological, biochemical, and molecular changes underlying the response of human bone marrow stromal cells to long-term simulated microgravity exposure during osteogenic differentiation. Our results show that osteogenic differentiation is reduced while energy metabolism is promoted. We found novel proteins were dysregulated under simulated microgravity, including CSC1-like protein, involved in the mechanotransduction of pressure signals, and PTPN11, SLC44A1 and MME which are involved in osteoblast differentiation pathways and which may become the focus of future translational projects. The investigation of cell proteome highlighted how simulated microgravity affects a relatively low number of proteins compared to time and/or osteogenic factors and has allowed us to reconstruct a hypothetical pipeline for cell response to simulated microgravity. Further investigation focused on the application of nanomaterials may help to increase understanding of how to treat or minimize the effects of microgravity.
 
Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, causes impaired adipocyte adhesion and defective adipose tissue architecture. Moreover, CTPS influences integrin distribution and dot-like deposition of type IV collagen (Col IV). Col IV-integrin signaling reciprocally regulates the assembly of cytoophidia in adipocytes. Our results demonstrate that a positive feedback signaling loop containing both cytoophidia and integrin adhesion complex couple tissue architecture and metabolism in Drosophila adipose tissue.
 
Preservation of blood vessel integrity, which is critical for normal physiology and organ function, is controlled at multiple levels, including endothelial junctions. However, the mechanism that controls the adequate assembly of endothelial cell junctions is not fully defined. Here, we uncover TAp73 transcription factor as a vascular architect that orchestrates transcriptional programs involved in cell junction establishment and developmental blood vessel morphogenesis and identify Angiomotin (AMOT) as a TAp73 direct transcriptional target. Knockdown of p73 in endothelial cells not only results in decreased Angiomotin expression and localization at intercellular junctions, but also affects its downstream function regarding Yes-associated protein (YAP) cytoplasmic sequestration upon cell–cell contact. Analysis of adherens junctional morphology after p73-knockdown in human endothelial cells revealed striking alterations, particularly a sharp increase in serrated junctions and actin bundles appearing as stress fibers, both features associated with enhanced barrier permeability. In turn, stabilization of Angiomotin levels rescued those junctional defects, confirming that TAp73 controls endothelial junction dynamics, at least in part, through the regulation of Angiomotin. The observed defects in monolayer integrity were linked to hyperpermeability and reduced transendothelial electric resistance. Moreover, p73-knockout retinas showed a defective sprout morphology coupled with hemorrhages, highlighting the physiological relevance of p73 regulation in the maintenance of vessel integrity in vivo. We propose a new model in which TAp73 acts as a vascular architect integrating transcriptional programs that will impinge with Angiomotin/YAP signaling to maintain junctional dynamics and integrity, while balancing endothelial cell rearrangements in angiogenic vessels.
 
The mechanisms of immunosuppressive function mediated by Treg cells. Treg cells secret the cytokines TGF-beta, IL-10 and IL-35 to directly inhibit the activation and proliferation of effector T cells. CTLA-4, LAG-3 and PD1 on Treg cells mediate the downregulation of APC cell functions, which prevents the activation of naïve T cells and effector T cells. CD25 expressed on Treg cells outcompetes IL-2 which is necessary for T cell in the peripheral to prevent its activation and proliferation. Adenosine produced by the hydrolyzation of CD39 and CD73 from ATP or ADP binds to A2A receptor on effector T cells, thereby inhibiting the proliferation and production of inflammatory cytokines. Treg cells also mediate the cytolysis of effector T cells by granzymes and perforin
Strategies to increase Treg cells frequencies and the suppressive function in vivo and in vitro. In vivo and in vitro strategies can be used to increase Treg cells number or function with immunomodulatory drugs. Low dose IL-2, engineered IL-2 muteins, the complex of IL-2/IL-2 receptor, IL-4, IL-5, IL-7, IL-12, IL-15, and IFN-γ are shown to have the potential to increase the activity of natural Treg cells or peripheral Treg cells against effector T cells in vivo. In addition, selective depletion of effector T cells by anti-CD3 antibodies can restore Treg cell predominance over effector T cells. Treg cells or naïve T cells isolated from the peripheral blood or the thymus of pediatric cardiac patients can be expanded and genetically modified in vitro for adaptive transfer to increase Treg cell numbers or improve specificity
Rheumatoid arthritis (RA) is an autoimmune disease that mainly affects the joints but also leads to systemic inflammation. Auto-reactivity and dysregulation of self-tolerance are thought to play a vital role in disease onset. In the pathogenesis of autoimmune diseases, disturbed immunosuppressive properties of regulatory T cells contribute to the dysregulation of immune homeostasis. In RA patients, the functions of Treg cells and their frequency are reduced. Therefore, focusing on the re-establishment of self-tolerance by increasing Treg cell frequencies and preventing a loss of function is a promising strategy for the treatment of RA. This approach could be especially beneficial for those patients who do not respond well to current therapies. In this review, we summarize and discuss the current knowledge about the function, differentiation and regulation of Treg cells in RA patients and in animal models of autoimmune arthritis. In addition, we highlight the therapeutic potential as well as the challenges of Treg cell targeting treatment strategies.
 
Optimizing conditions for staining mouse ASC, CASP8, and RIPK3. A–C Bone marrow-derived macrophages (BMDMs) from the indicated genotypes were fixed by 4% paraformaldehyde (PFA) or methanol (MeOH) and stained with indicated primary antibodies to evaluate staining specificity. Images are representative of at least three independent experiments
ASC specks are heterogenous in response to IAV infection. A–E Wild-type (WT) bone marrow-derived macrophages (BMDMs) were mock treated (A) or infected with influenza A virus (IAV) (B–E) and stained for ASC, RIPK3, and CASP8. Different compositions of ASC specks are shown. F ASC, RIPK3, and CASP8 staining of IAV-infected Zbp1–/– BMDMs. G Compositional analysis of ASC specks (n ≥ 100) in IAV-infected WT BMDMs at 12 h post-infection. Mean ± standard error is shown. Images are representative of at least three independent experiments
Inflammasome stimulation by LPS + ATP induces formation of ASC specks containing RIPK3 and CASP8. A Representative images of ASC specks from bone marrow-derived macrophages (BMDMs). Cells of the indicated genotypes were primed with 100 ng/μl LPS for 4 h, then stimulated with 5 mM ATP (i.e., LPS + ATP) for 10 min. B Expansion microscopy views of a RIPK3/CASP8-containing ring-shaped ASC speck induced by LPS + ATP. C Fluorescence intensity of ASC, RIPK3, and CASP8 in the ring section along the white arrow. X axis indicates the distance along the white arrow traveling from the base of the arrow to the arrowhead. D 3D view of the ASC speck in (B). Images are representative of at least three independent experiments
In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.
 
The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.
 
The ciliary body critically contributes to the ocular physiology with multiple responsibilities in the production of aqueous humor, vision accommodation and intraocular immunity. Comparatively little work, however, has revealed the single-cell molecular taxonomy of the human ciliary body required for studying these functionalities. In this study, we report a comprehensive atlas of the cellular and molecular components of human ciliary body as well as their interactions using single-cell RNA sequencing (scRNAseq). Cluster analysis of the transcriptome of 14,563 individual ciliary cells from the eyes of 3 human donors identified 14 distinct cell types, including the ciliary epithelium, smooth muscle, vascular endothelial cell, immune cell and other stromal cell populations. Cell-type discriminative gene markers were also revealed. Unique gene expression patterns essential for ciliary epithelium-mediated aqueous humor inflow and ciliary smooth muscle contractility were identified. Importantly, we discovered the transitional states that probably contribute to the transition of ciliary macrophage into retina microglia and verified no lymphatics in the ciliary body. Moreover, the utilization of CellPhoneDB allowed us to systemically infer cell–cell interactions among diverse ciliary cells including those that potentially participate in the pathogenesis of glaucoma and uveitis. Altogether, these new findings provide insights into the regulation of intraocular pressure, accommodation reflex and immune homeostasis under physiological and pathological conditions.
 
It has been reported that aging-generated gut microecosystem may promote host hepatic lipid dysmetabolism through shaping the pattern of secondary bile acids (BAs). Then as an oral drug, melatonin (Mel)-mediated beneficial efforts on the communication between gut microbiota and aging host are still not clearly. Here, we show that aging significantly shapes the pattern of gut microbiota and BAs, whereas Mel treatment reverses these phenotypes (P < 0.05), which is identified to depend on the existence of gut microbiota. Mechanistically, aging-triggered high-level expression of ileac farnesoid X receptor (FXR) is significantly decreased through Mel-mediated inhibition on Campylobacter jejuni (C. jejuni)-induced deconjugation of tauroursodeoxycholic acid (TUDCA) and glycoursodeoxycholic acid (GUDCA) (P < 0.05). The aging-induced high-level of serum taurine chenodeoxycholic acid (TCDCA) activate trimethylamine-N-oxide (TMAO)-triggered activating transcriptional factor 4 (ATF4) signaling via hepatic FXR, which further regulates hepatic BAs metabolism, whereas TUDCA inhibits aging-triggered high-level of hepatic ATF4. Overall, Mel reduces C. jejuni-mediated deconjugation of TUDCA to inhibit aging-triggered high-level expression of hepatic FXR, which further decreases hepatic TMAO production, to relieve hepatic lipid dysmetabolism.
 
CAPRIN1 is a ubiquitously expressed protein, abundant in the brain, where it regulates the transport and translation of mRNAs of genes involved in synaptic plasticity. Here we describe two unrelated children, who developed early-onset ataxia, dysarthria, cognitive decline and muscle weakness. Trio exome sequencing unraveled the identical de novo c.1535C > T (p.Pro512Leu) missense variant in CAPRIN1, affecting a highly conserved residue. In silico analyses predict an increased aggregation propensity of the mutated protein. Indeed, overexpressed CAPRIN1P512L forms insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders (ATXN2, GEMIN5, SNRNP200 and SNCA). Moreover, the CAPRIN1P512L mutation in isogenic iPSC-derived cortical neurons causes reduced neuronal activity and altered stress granule dynamics. Furthermore, nano-differential scanning fluorimetry reveals that CAPRIN1P512L aggregation is strongly enhanced by RNA in vitro. These findings associate the gain-of-function Pro512Leu mutation to early-onset ataxia and neurodegeneration, unveiling a critical residue of CAPRIN1 and a key role of RNA–protein interactions.
 
Theoretical energetic balance of ATP and heat productions in standard biochemical condition. Under biological operating conditions (pH = 7.5, I 0.2, T° = 37 °C), and considering a P/O ratio of 2.5 per NADH and 1.5 per FADH2, the degradation of one molecule of glucose (2871 kJ) successively through the glycolysis in the cytoplasm, the tri-carboxylic acid cycle, and the oxidative phosphorylation in mitochondria produces. A 32 ATP (1152 kJ ≈40%), including 28 OXPHOS-generated ATP and 1719 kJ (≈60%) of heat. B 25 ATP (900 kJ≈30%), including 21 OXPHOS-generated ATP and 1971 kJ (≈70%) of heat are produced from glucose oxidation when the mechanistic P/O ratios of NADH and FADH2 are corrected for uncoupling mechanisms, considering a physiological efficiency of 75–80%. These values illustrate the major role of mitochondria in heat production linked to the energetic metabolism
Mitochondrial energetic and thermogenesis balance. In mitochondria, substrate oxidation energy is converted into ATP and heat. The maximal global efficiency of OXPHOS is ~ 40%/60% for ATP/Heat. However, in resting rat muscle, H⁺ leak (detailed in section II.) could represent ≈50% of O2 consumption. In this case, the P/O is reduced by an half and the ATP/Heat balance increases up to 20%/80%. According to cellular needs, ATP/heat balance in mitochondria can turn to either situations: A An uncoupling activity with increased oxidation rate. Due to the uncoupling mechanisms, the ATP/Heat balance is modulated in favor of heat production, which is preeminent in BAT through UCP1 activation, and in other tissues as skeletal muscle and liver, through UCP1-independent mechanisms, such as increasing ANT activity. B At the maximum phosphorylating activity, the H⁺ leak represents an insignificant proportion of O2 consumption. The ATP/Heat balance is close to the theoretical maximal efficiency of 40%/60%. However, increased oxidation rate results in a rise in ATP synthesis and a consecutive heat production, a process common to all tissues.
OXPHOS uncoupling mechanisms promote mitochondrial heat production. Mitochondrial uncoupling promotes heat production by reducing the ATP production efficiency. Uncoupling Protein 1 (UCP1) activation induces H⁺ leak, which dissipates the Δp thereby uncoupling substrate oxidation from ATP synthesis while increasing thermogenesis. The Adenine Nucleotide Translocator (ANT), in addition to its main function as ATP/ADP carrier, can also act as H⁺ transporter. ANT-mediated H⁺ leak requires protonatable Free Fatty Acids (FFA), which would act as cofactors. FFA-dependent H⁺ leak competes with nucleotide exchange activity in the ANT translocation pathway, switching from H⁺ leak, promoting heat production, to ATP/ADP translocation, promoting ATP production. e⁻ slip at CIV and H⁺ slip at ATP synthase could lower the P/O ratio. ROS production through e⁻ leak deflects electron transfer occurring from NADH (CI) or FADH2 (CIII) oxidations. The glycerol-3-phosphate (G3P) shuttle catalyzes an apparent exchange of cytosolic NADH for mitochondrial FADH2, directly transferring electrons to CIII via the ubiquinone. This reduces the H⁺/O2 stoichiometry from 10H⁺/2e⁻ for NADH to 6H⁺/2e⁻ [23], ultimately reducing the P/O stoichiometry and the efficiency of ATP production.
Mitochondrial respiration rate regulators impacting mitochondrial heat production. ATP-dependent futile cycles promote high ATP turnover, thereby increasing OXPHOS rate and inherent energy loss as heat. Creatine Kinases (CK) connect the ATP consumption to ATP-production sites through PhosphoCreatine (PCr)/Creatine (Cr) shuttle. Within mitochondria, mtCK in the intermembrane space is coupled to ADP phosphorylation through ANT, maintaining high ADP/ATP ratio within the mitochondrial matrix. Futile cycle of creatine dephosphorylation drives ATP hydrolysis, consequently increasing OXPHOS rate. Ca²⁺ cycling by SERCA in reticulum endoplasmic increases both ATP hydrolysis and Ca²⁺ accumulation in the cytosol. ATP hydrolysis promotes mitochondrial ATP re-synthesis while high substrate oxidation flux is sustained thanks to Ca²⁺ accumulation in mitochondria through mitochondrial calcium uniporter (MCU). Metabolic energy sensors, such as SIRT3, can also stimulate substrate supply and respiratory chain activity, by modulating the activity of TCA cycle enzymes and respiratory chain complexes.
Understanding temperature production and regulation in endotherm organisms becomes a crucial challenge facing the increased frequency and intensity of heat strokes related to global warming. Mitochondria, located at the crossroad of metabolism, respiration, Ca2+ homeostasis, and apoptosis, were recently proposed to further act as cellular radiators, with an estimated inner temperature reaching 50°C in common cell lines. This inner thermogenesis might be further exacerbated in organs devoted to produce consistent efforts as muscles, or heat as brown adipose tissue, in response to acute solicitations. Consequently, pathways promoting respiratory chain uncoupling and mitochondrial activity, such as Ca2+ fluxes, uncoupling proteins, futile cycling, and substrate supplies, provide the main processes controlling heat production and cell temperature. The mitochondrial thermogenesis might be further amplified by cytoplasmic mechanisms promoting the over-consumption of ATP pools. Considering these new thermic paradigms, we discuss here all conventional wisdoms linking mitochondrial functions to cellular thermogenesis in different physiological conditions.
 
Endometrial cancer (EC) is the most common type of gynecologic cancer in women of developed countries. Despite surgery combined with chemo-/radiotherapy regimens, overall survival of patients with high-risk EC tumors is poor, indicating a need for novel therapies. The MEK5-ERK5 pathway is activated in response to growth factors and to different stressors, including oxidative stress and cytokines. Previous evidence supports a role for the MEK5-ERK5 pathway in the pathology of several cancers. We investigated the role of ERK5 in EC. In silico analysis of the PanCancer Atlas dataset showed alterations in components of the MEK5-ERK5 pathway in 48% of EC patients. Here, we show that ERK5 inhibition or silencing decreased EGF-induced EC cell proliferation, and that genetic deletion of MEK5 resulted in EC impaired proliferation and reduced tumor growth capacity in nude mice. Pharmacologic inhibition or ERK5 silencing impaired NF-kB pathway in EC cells and xenografts. Furthermore, we found a positive correlation between ERK5 and p65/RELA protein levels in human EC tumor samples. Mechanistically, genetic or pharmacologic impairment of ERK5 resulted in downregulation of NEMO/IKKγ expression, leading to impaired p65/RELA activity and to apoptosis in EC cells and xenografts, which was rescued by NEMO/IKKγ overexpression. Notably, ERK5 inhibition, MEK5 deletion or NF-kB inhibition sensitized EC cells to standard EC chemotherapy (paclitaxel/carboplatin) toxicity, whereas ERK5 inhibition synergized with paclitaxel to reduce tumor xenograft growth in mice. Together, our results suggest that the ERK5-NEMO-NF-κB pathway mediates EC cell proliferation and survival. We propose the ERK5/NF-κB axis as new target for EC treatment.
 
Intestinal stem cells (ISCs) decode and coordinate various types of nutritional information from the diet to support the crypt–villus axis architecture, but how specific dietary molecules affect intestinal epithelial homeostasis remains unclear. In the current study, L-glutamate (Glu) supplementation in either a nitrogen-free diet (NFD) or a corn-soybean meal diet (CSMD) stimulated gut growth and ISC expansion in weaned piglets. Quantitative proteomics screening identified the canonical Wnt signalling pathway as a central regulator of intestinal epithelial development and ISC activity in vivo. Importantly, the Wnt transmembrane receptor Frizzled7 (FZD7) was upregulated in response to dietary Glu patterns, and its perturbations in intestinal organoids (IOs) treated with a specific inhibitor and in FZD7-KO IPEC-J2 cells disrupted the link between Glu inputs and β-catenin signalling and a subsequent reduction in cell viability. Furthermore, co-localization, coimmunoprecipitation (Co-IP), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST) revealed that Glu served as a signalling molecule directly bound to FZD7. We propose that FZD7-mediated integration of the extracellular Glu signal controls ISC proliferation and differentiation, which provides new insights into the crosstalk of nutrients and ISCs.
 
Schematic structure of the human SOX9 protein. The dimerization domain (DIM) precedes the HMG box. Two transactivation domains are located in the middle (TAM) and at the C-terminus (TAC). The proline, glutamine and alanine (PQA)-rich domain is required for transactivation. Phosphorylation of serine (S) residues, acetylation and sumoylation of lysine (K) residues are highlighted
Regulation of target genes by SOX9 and partners. A SOX9 homodimer binds to inverted SOX binding sites in multiple enhancers of chondrocyte-specific genes. It physically interacts with SOX5/SOX6 and other partners which bind to DNA sequences close to SOX9 binding sites. SRY and SF1 function together to regulate Amh and Sox9 itself in Sertoli cells. B Apart from transactivating targets, SOX9 forms complex with GLI factors to repress genes such as Col10a1 and Vegfa. C SOX9 and partner proteins regulate stepwise progression of tissue development. In Sertoli cells, SRY and SF1 induce Sox9 expression and then SOX9 forms complex with SF1 to promote subsequent gene expression. During astrocyte differentiation, SOX9 induces Nfia expression and then interacts with NFIA protein to promote astrocytic gene expression. D Multiple TFs bind to super-enhancers to regulate cartilage-specific gene expression in chondrocytes, while SOX9 indirectly engages the genome via interacting with basal transcriptional components to regulate widely expressed gene expression in chondrocytes
The transcription factor SOX9 is essential for the development of multiple organs including bone, testis, heart, lung, pancreas, intestine and nervous system. Mutations in the human SOX9 gene led to campomelic dysplasia, a haploinsufficiency disorder with several skeletal malformations frequently accompanied by 46, XY sex reversal. The mechanisms underlying the diverse SOX9 functions during organ development including its post-translational modifications, the availability of binding partners, and tissue-specific accessibility to target gene chromatin. Here we summarize the expression, activities, and downstream target genes of SOX9 in molecular genetic pathways essential for organ development, maintenance, and function. We also provide an insight into understanding the mechanisms that regulate the versatile roles of SOX9 in different organs.
 
Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an extremely variable clinical course. We have recently shown that high catalase (CAT) expression identifies patients with an aggressive clinical course. Elucidating mechanisms regulating CAT expression in CLL is preeminent to understand disease mechanisms and develop strategies for improving its clinical management. In this study, we investigated the role of the CAT promoter rs1001179 single nucleotide polymorphism (SNP) and of the CpG Island II methylation encompassing this SNP in the regulation of CAT expression in CLL. Leukemic cells harboring the rs1001179 SNP T allele exhibited a significantly higher CAT expression compared with cells bearing the CC genotype. CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β transcription factors. Moreover, CLL cells exhibited lower methylation levels than normal B cells, in line with the higher CAT mRNA and protein expressed by CLL in comparison with normal B cells. Methylation levels at specific CpG sites negatively correlated with CAT levels in CLL cells. Inhibition of methyltransferase activity induced a significant increase in CAT levels, thus functionally validating the role of CpG methylation in regulating CAT expression in CLL. Finally, the CT/TT genotypes were associated with lower methylation and higher CAT levels, suggesting that the rs1001179 T allele and CpG methylation may interact in regulating CAT expression in CLL. This study identifies genetic and epigenetic mechanisms underlying differential expression of CAT, which could be of crucial relevance for the development of therapies targeting redox regulatory pathways in CLL. Graphical abstract
 
The cytoprotective ATP receptor P2Y11 is upregulated during M2 macrophage differentiation and contributes to the anti-inflammatory properties of this macrophage subset. Here, we studied P2Y11-induced reprogramming of human M2 macrophages at the level of mRNA and protein expression. Upregulation of IL-1 receptor (IL-1R) and its known downstream effectors VEGF, CCL20 and SOCS3 as well as downregulation of the ATP-degrading ecto-ATPase CD39 emerged as hallmarks of P2Y11 activation. The anti-inflammatory signature of the P2Y11 transcriptome was further characterized by the downregulation of P2RX7, toll-like receptors and inflammasome components. P2Y11-induced IL-1R upregulation formed the basis for reinforced IL-1 responsiveness of activated M2 macrophages, as IL-1α and IL-1ß each enhanced P2Y11-induced secretion of VEGF and CCL20 as well as the previously reported shedding of soluble tumor necrosis factor receptor 2 (sTNFR2). Raising intracellular cyclic AMP (cAMP) in M2 macrophages through phosphodiesterase 4 inhibition enhanced P2Y11-driven responses. The cAMP-binding effector, exchange protein activated by cAMP 1 (Epac1), which is known to induce SOCS3, differentially regulated the P2Y11/IL-1R response because pharmacological Epac1 inhibition enhanced sTNFR2 and CCL20 release, but had no effect on VEGF secretion. In addition to cAMP, calcium and protein kinase C participated in P2Y11 signaling. Our study reveals how P2Y11 harnesses canonical and IL-1R signaling to promote an anti-inflammatory and pro-angiogenic switch of human M2 macrophages, which may be controlled in part by an Epac1-SOCS3 axis.
 
In our search for innovative drugs that could improve periodontal treatment outcomes, autophagy and its anomalies represent a potential target for therapeutic intervention. We sought to identify autophagy defects in murine experimental periodontitis and study the effectiveness of P140, a phosphopeptide known to bind HSPA8 and inhibit its chaperone properties, and that corrects autophagy dysfunctions in several autoimmune and inflammatory diseases. Experimental periodontitis was induced by placing silk ligature around mandibular first molars. Sick mice were treated intraperitoneally with either P140 or a control, scrambled peptide. After 10 days, mandibles were harvested and bone loss was measured by micro-CT. Immune cells infiltration was studied by histological analyses. Cytokines levels and autophagy-related markers expression were evaluated by qRT-PCR and western blotting. A comparison with non-affected mice revealed significant alterations in the autophagy processes in mandibles of diseased mice, especially in the expression of sequestosome 1/p62, Maplc3b, Atg5, Ulk1, and Lamp2. In vivo, we showed that P140 normalized the dysregulated expression of several autophagy-related genes. In addition, it diminished the infiltration of activated lymphocytes and pro-inflammatory cytokines. Unexpectedly P140 decreased the extent of bone loss affecting the furcation and alveolar areas. Our results indicate that P140, which was safe in clinical trials including hundreds of autoimmune patients with systemic lupus erythematosus, not only decreases the inflammatory effects observed in mandibular tissues of ligation-induced mice but strikingly also contributes to bone preservation. Therefore, the therapeutic peptide P140 could be repositioned as a decisive breakthrough for the future therapeutic management of periodontitis.
 
Activation of the Ras signaling pathway promotes the growth of malignant human glioblastoma multiforme (GBM). Mutations in Ras are rare in GBM, elevated levels of activated Ras are prevalently observed in GBM. However, the potential mechanism of how Ras is activated in GBM remains unclear. In this study, we screened a new interacted protein of Ras, PHLDA1. Our findings confirmed that PHLDA1 acted as an oncogene and promoted glioma progression and recurrence. We demonstrated that PHLDA1 was upregulated in GBM tissues and cells. PHLDA1 overexpression promoted cell proliferation and tumor growth. In terms of mechanism, PHLDA1 promoted cell proliferation by regulating Ras/Raf/Mek/Erk signaling pathway. Moreover, Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation. PHLDA1 and Src competed for binding with Ras, inhibiting Ras phosphorylation by Src and rescuing Ras activity. This study may provide a new idea of the molecular mechanism underlying glioma progression and a novel potential therapeutic target for comprehensive glioblastoma treatment.
 
Diabetic nephropathy (DN) is a significant complication of diabetes and the leading cause of end-stage renal disease. Hyperglycemia-induced dysfunction of the glomerular podocytes is a major contributor to the deterioration of renal function in DN. Previously, we demonstrated that podocyte-specific disruption of the Src homology phosphatase 2 (Shp2) ameliorated lipopolysaccharide-induced renal injury. This study aims to evaluate the contribution of Shp2 to podocyte function under hyperglycemia and explore the molecular underpinnings. We report elevated Shp2 in the E11 podocyte cell line under high glucose and the kidney under streptozotocin- and high-fat diet-induced hyperglycemia. Consistently, Shp2 disruption in podocytes was associated with partial renoprotective effects under hyperglycemia, as evidenced by the preserved renal function. At the molecular level, Shp2 deficiency was associated with altered renal insulin signaling and diminished hyperglycemia-induced renal endoplasmic reticulum stress, inflammation, and fibrosis. Additionally, Shp2 knockdown in E11 podocytes mimicked the in vivo deficiency of this phosphatase and ameliorated the deleterious impact of high glucose, whereas Shp2 reconstitution reversed these effects. Moreover, Shp2 deficiency attenuated high glucose-induced E11 podocyte migration. Further, we identified the protein tyrosine kinase FYN as a putative mediator of Shp2 signaling in podocytes under high glucose. Collectively, these findings suggest that Shp2 inactivation may afford protection to podocytes under hyperglycemia and highlight this phosphatase as a potential target to ameliorate glomerular dysfunction in DN.
 
OPA1 deficiency repressed oxidative glucose metabolism. A Mito-tracker green of OPA1-wild-type (OPA1-WT) and OPA1-knockout (OPA1-KO) MEFs. B An illustration of the metabolic flow of oxidative glucose metabolism in [U-¹³C]glucose cultured cells. The empty dots represent the natural carbon 12, and the solid red dots represent the carbon 13 isotope. The full six-carbon-labeled (¹³C) glucose produces three-carbon-labeled pyruvate (m + 3), which enters the TCA cycle to produce two-carbon (m + 2) labeled acetyl-CoA, citrate, α-ketoglutarate (α-KG), succinate, fumarate, and malate. The m + 2 labeled citrate further produces carbon-labeled palmitate. C Mass isotopologue analysis of citrate, α-KG, and malate in OPA1-WT and OPA1-KO MEFs cultured with [U-¹³C]glucose and unlabeled glutamine for 4 h. D The relative abundances of citrate, α-KG, and malate in OPA1-WT and OPA1-KO MEFs cultured with [U-¹³C]glucose and unlabeled glutamine for 4 h (n = 3 cultures from a representative experiment). The data in C, D were analyzed by multiple t-test and the significant level was set as *p < 0.05 (OPA1-KO vs. OPA1-WT). The experiments were repeated at least three times and n = 3 cultures from a representative experiment
OPA1 deficiency induced glutamine-dependent reductive carboxylation. A An illustration of the metabolic flow of reductive glutamine metabolism in [U-¹³C]glutamine cultured cells. The empty dots represent the natural carbon 12, and the solid red and gray dots represent the carbon 13 isotope. The full five-carbon-labeled (¹³C) glutamine produces five-carbon-labeled α-ketoglutarate (a-KG) (m + 5), which further produces five-carbon-labeled citrate (m + 5) by mitochondrial IDH2 and cytosolic IDH1. After the release of two-carbon-labeled acetyl-CoA, the five-carbon-labeled citrate would generate one molecule of two-carbon-labeled acetyl-CoA for palmitate synthesis and another molecule of cytosolic three-carbon-labeled oxaloacetate (m + 3) as well as malate (m + 3) and fumarate (m + 3). Mass isotopologue analysis of citrate (B) and the other three TCA cycle intermediates including α-KG, fumarate, and malate (C) in OPA1-WT and OPA1-KO MEFs cultured with [U ¹³C]glutamine and unlabeled glucose for 4 h. D Mass isotopologue analysis of citrate and palmitate as well as the relative contribution of glucose oxidation/glutamine reduction to lipogenic AcCoA in OPA1-WT and OPA1-KO MEFs cultured with [U-¹³C]glucose and unlabeled glutamine for 20 h or cultured with [U-¹³C]glutamine and unlabeled glutamine for 20 h. The data in 2B–D were analyzed by multiple t-test and the significant level was set as *p < 0.05 (OPA1-KO vs. OPA1-WT). The experiments were repeated at least three times and n = 3 cultures from a representative experiment
The modeling of metabolic fluxes in OPA1-deficient MEFs. A The comparative modeling of metabolic fluxes in OPA1-WT and OPA1-KO MEFs. See Table S2 for definitions of quantitative flux values. The color scale reflects the ratio of each flux in OPA1-KO cells relative to OPA1-WT controls. B The immunoblots of OPA1, LDH, PDH, MPC1/2, CTP, DIC, IDH1/2/3A, and FASN in OPA1-WT and OPA1-KO MEFs. β-actin acts as the internal protein. LDH represents lactate dehydrogenase, PDH represents pyruvate dehydrogenase, MPC1/2 represents mitochondrial pyruvate carrier, CTP represents citrate transport protein, DIC represents mitochondrial dicarboxylate carrier, IDH1/2/3A represent isocitrate dehydrogenases, and FASN represents fatty acid synthase
OPA1-deficient MEFs rely on cytosolic reductive carboxylation for growth. A Relative live cell number of OPA1-WT and OPA1-KO MEFs after treatment with the medium containing DMSO and 5 µM IDHi for 48 h (vs. OPA1-WT-DMSO). The live cells were counted with Trypan blue staining. The data were analyzed by multiple t-test and the significant level was set as *p < 0.05 (IDH1i vs. DMSO) in each cell line. The experiments were repeated at least three times and n = 3 cultures from a representative experiment. Mass isotopologue analysis of palmitate (B), citrate and malate (C) in OPA1-KO MEFs cultured with [U-¹³C]glutamine and unlabeled glucose medium containing DMSO or 5 µM IDH1 inhibitor (IDHi, GSK321) for 20 h. The data were analyzed by multiple t-test and the significant level was set as *p < 0.05 (IDH1i vs. DMSO) in each cell line. The experiments were repeated at least three times and n = 3 cultures from a representative experiment
The schematic model of OPA1-deficiency-induced glutamine-dependent reductive carboxylation for DNL that is essential for cell growth
OPA1, a dynamin-related GTPase mutated in autosomal dominant optic atrophy, is essential for the fusion of the inner mitochondrial membrane. Although OPA1 deficiency leads to impaired mitochondrial morphology, the role of OPA1 in central carbon metabolism remains unclear. Here, we aim to explore the functional role and metabolic mechanism of OPA1 in cell fitness beyond the control of mitochondrial fusion. We applied [U-¹³C]glucose and [U-¹³C]glutamine isotope tracing techniques to OPA1-knockout (OPA1-KO) mouse embryonic fibroblasts (MEFs) compared to OPA1 wild-type (OPA1-WT) controls. Furthermore, the resulting tracing data were integrated by metabolic flux analysis to understand the underlying metabolic mechanism through which OPA1 deficiency reprograms cellular metabolism. OPA1-deficient MEFs were depleted of intracellular citrate, which was consistent with the decreased oxygen consumption rate in these cells with mitochondrial fission that is not balanced by mitochondrial fusion. Whereas oxidative glucose metabolism was impaired, OPA1-deficient cells activated glutamine-dependent reductive carboxylation and subsequently relied on this reductive metabolism to produce cytosolic citrate as a predominant acetyl-CoA source for de novo fatty acid synthesis. Prevention of cytosolic glutamine reductive carboxylation by GSK321, an inhibitor of isocitrate dehydrogenase 1 (IDH1), largely repressed lipid synthesis and blocked cell proliferation in OPA1-deficient MEFs. Our data support that, when glucose oxidation failed to support lipogenesis and proliferation in cells with unbalanced mitochondrial fission, OPA1 deficiency stimulated metabolic anaplerosis into glutamine-dependent reductive carboxylation in an IDH1-mediated manner.
 
The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/– mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.
 
Foamy macrophages and microglia containing lipid droplets (LDs) are a pathological hallmark of demyelinating disorders affecting the central nervous system (CNS). We and others showed that excessive accumulation of intracellular lipids drives these phagocytes towards a more inflammatory phenotype, thereby limiting CNS repair. To date, however, the mechanisms underlying LD biogenesis and breakdown in lipid-engorged phagocytes in the CNS, as well as their impact on foamy phagocyte biology and lesion progression, remain poorly understood. Here, we provide evidence that LD-associated protein perilipin-2 (PLIN2) controls LD metabolism in myelin-containing phagocytes. We show that PLIN2 protects LDs from lipolysis-mediated degradation, thereby impairing intracellular processing of myelin-derived lipids in phagocytes. Accordingly, loss of Plin2 stimulates LD turnover in foamy phagocytes, driving them towards a less inflammatory phenotype. Importantly, Plin2-deficiency markedly improves remyelination in the ex vivo brain slice model and in the in vivo cuprizone-induced demyelination model. In summary, we identify PLIN2 as a novel therapeutic target to prevent the pathogenic accumulation of LDs in foamy phagocytes and to stimulate remyelination.
 
The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.
 
To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine–chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.
 
Schematic representation of antibody formats used in immunocytokines. Variable domain light chain (VL), constant region light chain (CL), variable domain heavy chain (VH), constant region heavy chain (CH), immunoglobulin G (IgG), antigen-binding fragment (F(ab′)), single chain variable fragment (scFv), fragment crystallizable region (Fc)
Schematic representation of immunocytokine formats in (pre-)clinical trials
Schematic representation of bispecific antibody formats. Abbreviations used: variable domain light chain (VL), constant region light chain (CL), variable domain heavy chain (VH), constant region heavy chain (CH). Bispecific T cell Engagers (BiTEs), Dual-Affinity Re-targeting proteins (DARTs)
Schematic overview of the prevention of payload-mediated toxicity via the separation of an active TNF-homotrimer into inactive TNF-monomers. This allows the formation of the active homotrimer at sites where the antibodies aggregate, i.e. the tumor micromilieu
Monoclonal antibody (mAb) therapy has successfully been introduced as treatment of several lymphomas and leukemias. However, solid tumors reduce the efficacy of mAb therapy because of an immune-suppressive tumor micro-environment (TME), which hampers activation of effector immune cells. Pro-inflammatory cytokine therapy may counteract immune suppression in the TME and increase mAb efficacy, but untargeted pro-inflammatory cytokine therapy is limited by severe off-target toxicity and a short half-life of cytokines. Antibody-cytokine fusion proteins, also referred to as immunocytokines, provide a solution to either issue, as the antibody both acts as local delivery platform and increases half-life. The antibody can furthermore bridge local cytotoxic immune cells, like macrophages and natural killer cells with tumor cells, which can be eliminated after effector cells are activated via the cytokine. Currently, a variety of different antibody formats as well as a handful of cytokine payloads are used to generate immunocytokines. However, many potential formats and payloads are still left unexplored. In this review, we describe current antibody formats and cytokine moieties that are used for the development of immunocytokines, and highlight several immunocytokines in (pre-)clinical studies. Furthermore, potential future routes of development are proposed.
 
The proposed model of dynamic microglial phenotypes during the development of neurodegenerative diseases. The initial effects of microglial activation could be beneficial in clearing misfolded protein aggregates and cellular debris. However, persistent and chronic overactivation of microglia triggered by these aggregates, sustained immune stimuli, or the aging process contributes critically to the development of neurodegenerative processes. During immunopathogenesis, microglial activation gradually switches from a beneficial to a detrimental phenotype, leading to extensive neuronal death. Interfering with the microglial phenotype promises to halt chronic immunopathogenesis and create a more balanced immune milieu in the CNS
Heterogeneity of microglia in neurodegenerative diseases. Harmful microglial phenotypes usually release various pro-inflammatory factors, and cause extensive neuronal death, eventually leading to cognitive deficits, memory impairment and movement disorders. MGnD is essentially induced by apoptotic neurons and plays a pro-inflammatory role in neuronal injury. LDAM is characterized by the accumulation of lipid droplets specifically in aging microglia and releases pro-inflammatory cytokines that exacerbate neuronal death. In contrast, beneficial microglial phenotypes generally induce multiple anti-inflammatory factors and enhance phagocytosis of cellular debris or protein aggregates, to promote neuronal survival and improve memory and cognitive function. PAM may be a neuroprotective phenotype that can physically prevent Aβ deposition. WAM appears to play an anti-inflammatory role that may enhance the phagocytosis of microglia. DAM was discovered by single-cell RNA sequencing specifically in microglia adjacent to amyloid deposits in AD. It is thought to be neuroprotective by enhancing phagocytosis, but may also be neurotoxic by upregulating AD risk genes and enhancing the APOE-TREM2 signaling in late-stage AD, which has been shown to be detrimental in neurodegenerative diseases
DNA modification and histone modification during neurodegeneration. A DNA modification in microglia. SIRT1 and 5-azacytide (5-aza) inhibit DNMT1, increasing the expression of IL-1β, thereby exacerbating pro-inflammation in microglia. TET2 oxidizes 5-mC to 5-hmC, increasing the release of various pro-inflammatory cytokines. B Histone modification in microglia. PRC2 is a histone methylase complex that inhibits the expression of clearance genes such as AXL, MS4A7, and ADAMTS18, thereby impairing phagocytosis of microglia. JMJD3 as a histone demethylase could induce the expression of ARG1 and in turn suppress the production of pro-inflammatory iNOS, IL-1β, and IL-6, to attenuate microglia-induced neuroinflammation. Moreover, VPA and CAY10683 inhibit HDACs to decrease the expression of IL-1β and TNF-α to attenuate neuroinflammation. Histone phosphorylation is also an important epigenetic modulation in microglia. The elevated histone H3S10phK14ac level can increase the expression of pro-inflammatory genes such as IL-1β, IL-6, TNF-α, iNOS, and c-Fos in activated microglia. Histone lactylation, as an emerging epigenetic modification, was increased in the brain of both 5xFAD mice and patients with AD. The elevated level of H4K12la was specifically detected in Aβ plaque-adjacent microglia, which composed the glycolysis/H4K12la/glycolytic genes positive feedback loop that aggravates microglial dysfunction in AD. Particularly, H4K12la modification is enriched at the promoters of glycolytic genes, such as PKM2, HIF-1α and LDHA, and further activates their transcription and increases glycolytic activity. PEP: phosphoenolpyruvate
RNA modification and non-coding RNA regulation in macrophage/microglia. A m6A modification. METTL3 methylates STAT1 mRNA to increase its stability, thereby increasing STAT1 expression and exacerbating pro- inflammation. In contrast, FTO deficiency could decrease the phosphorylation levels of IKKα/β, IκBα, and p65, which underlie NF-κB signaling, thereby increasing the production of pro-inflammatory cytokines. B Non-coding RNA regulations. LncRNAs function by binding to RBPs or serving as miRNA sponges to regulate gene expression. Lnc-p21 is induced by LPS and could sequester miR-181/PKC-δ to promote NF-κB signaling and increase the release of pro-inflammatory cytokines. The lnc-MALAT1 interacts with EZH2 to suppress the expression of NRF2, which, in turn, inhibits the release of ROS. The lnc-GAS5 binds to PRC2 to suppress the expression of IRF4, thereby reducing the activation of M2 microglia. Lnc-Nostrill is physically associated with NF-kB subunit p65 for the induction of iNOS expression and NO production. Lnc-SNHG1 acted as a competing endogenous RNA for miR-7 to promote NLRP3 expression and activated NLRP3 inflammasome. circRNAs have similar properties to lncRNAs in terms of their mechanisms of action as miRNA sponges or RBP-binding docks. circ_0000518 serves as the scaffold for binding with FUS protein. miRNAs have been extensively studied as pro-inflammatory or anti-inflammatory in microglia. They act by binding to the 3’UTR of target mRNAs to destabilize and knockdown transcripts, and by binding to lncRNAs or circRNAs. MiR-124, miR-146a and miR-21 can bind with downstream genes to eventually suppress the release of pro-inflammatory cytokines, while miR-155 basically promotes the expression of pro-inflammatory genes. Note that non-coding RNAs could be packaged into EVs that communicate between cells and could also be used as important therapeutic vehicles
Perspectives in studying microglial biology. Emerging studies have shown that several key factors such as genetic variations, gut microbiota, and aging may modulate microglial activation with different mechanisms likely controlled by epigenetic regulations. (i) The gut microbiota is required for the maintenance of proper microglial properties and is likely involved in neuroinflammation-induced diseases. (ii) Using high-throughput sequencing techniques such as GWAS and multi-omics analysis numerous risk genes associated with neurodegenerative diseases have been detected selectively or preferentially in microglia rather than neurons in large populations of diseased and healthy patients. It is suggested that risk variations in microglia may alter the microglial phenotype and immune profile per se and eventually lead to chronic microglia overactivation and the development of neurodegenerative diseases. (iii) To circumvent the differences between mouse and human microglia, novel methods have been developed to differentiate wild-type hPSCs, as well as isogenic hPSCs edited with CRISPR/Cas9 into microglia that can provide abundant mature microglia from humans, especially patients, for disease modeling and studying the mechanisms of microglial activation
Microglia are resident immune cells in the brain and play a central role in the development and surveillance of the nervous system. Extensive gliosis is a common pathological feature of several neurodegenerative diseases, such as Alzheimer's disease (AD), the most common cause of dementia. Microglia can respond to multiple inflammatory insults and later transform into different phenotypes, such as pro- and anti-inflammatory phenotypes, thereby exerting different functions. In recent years, an increasing number of studies based on both traditional bulk sequencing and novel single-cell/nuclear sequencing and multi-omics analysis, have shown that microglial phenotypes are highly heterogeneous and dynamic, depending on the severity and stage of the disease as well as the particular inflammatory milieu. Thus, redirecting microglial activation to beneficial and neuroprotective phenotypes promises to halt the progression of neurodegenerative diseases. To this end, an increasing number of studies have focused on unraveling heterogeneous microglial phenotypes and their underlying molecular mechanisms, including those due to epigenetic and non-coding RNA modulations. In this review, we summarize the epigenetic mechanisms in the form of DNA and histone modifications, as well as the general non-coding RNA regulations that modulate microglial activation during immunopathogenesis of neurodegenerative diseases and discuss promising research approaches in the microglial era.
 
Oncosis (from Greek ónkos , meaning “swelling”) is a non-apoptotic cell death process related to energy depletion. In contrast to apoptosis, which is the main form of cell death induced by anticancer drugs, oncosis has been relatively less explored but holds potential to overcome drug resistance phenomena. In this study, we report a novel rationally designed mitochondria-targeted iridium(III) complex ( OncoIr3 ) with advantageous properties as a bioimaging agent. OncoIr3 exhibited potent anticancer activity in vitro against cancer cells and displayed low toxicity to normal dividing cells. Flow cytometry and fluorescence-based assays confirmed an apoptosis-independent mechanism involving energy depletion, mitochondrial dysfunction and cellular swelling that matched with the oncotic process. Furthermore, a Caenorhabditis elegans tumoral model was developed to test this compound in vivo, which allowed us to prove a strong oncosis-derived antitumor activity in animals (with a 41% reduction of tumor area). Indeed, OncoIr3 was non-toxic to the nematodes and extended their mean lifespan by 18%. Altogether, these findings might shed new light on the development of anticancer metallodrugs with non-conventional modes of action such as oncosis, which could be of particular interest for the treatment of apoptosis-resistant cancers. Graphical abstract
 
Human space travel and exploration are of interest to both the industrial and scientific community. However, there are many adverse effects of spaceflight on human physiology. In particular, there is a lack of understanding of the extent to which microgravity affects the immune system. T cells, key players of the adaptive immune system and long-term immunity, are present not only in blood circulation but also reside within the tissue. As of yet, studies investigating the effects of micro-gravity on T cells are limited to peripheral blood or traditional 2D cell culture that recapitulates circulating blood. To better mimic interstitial tissue, 3D cell culture has been well established for physiologically and pathologically relevant models. In this work, we utilize 2D cell culture and 3D collagen matrices to gain an understanding of how simulated microgravity, using a random positioning machine, affects both circulating and tissue-resident T cells. T cells were studied in both resting and activated stages. We found that 3D cell culture attenuates the effects of simulated microgravity on the T cells transcriptome and nuclear irregularities compared to 2D cell culture. Interestingly, simulated microgravity appears to have less effect on activated T cells compared to those in the resting stage. Overall, our work provides novel insights into the effects of simulated microgravity on circulating and tissue-resident T cells which could provide benefits for the health of space travellers.
 
Zebrafish as a vertebrate model for scoliosis. Left: Different types of scoliosis and their potential causes from zebrafish studies. Scoliosis due to neuromuscular defects is also illustrated as CS like group. Asterisks indicate the abnormally developed vertebrae in CS-like zebrafish mutants. Right: Various bone staining and imaging methods used to evaluate skeleton development in zebrafish. At larvae stages, the notochord and vacuoles can be easily visualized via bright-field image (top left) or LysoTracker dye staining (top middle). Skeletal development in zebrafish can be visualized via alcian blue-Alizarin red double staining (top right). At juvenile or adult stages, skeleton development can be visualized via transgenic labeling, calcein staining, Alizarin red staining or micro-CT
Phenotypes of CS-like and IS-like zebrafish mutants. Left, Micro-CT images showing the morphology of spine in wild type, dstyk mutant (CS-like) and uts2r3 mutant (IS-like). Alizarin red staining images of the vertebral body in different mutants were shown on the right. Arrow indicates the fusion between two adjacent vertebrae in dstyk mutants. Images of dstyk mutants courtesy of Xianding Sun and Lin Chen
Model illustrating the Urotensin signaling pathway during body axis development of zebrafish. The beating of ependymal motile cilia drives CSF flow, which mediates the transmission of epinephrine signals and the assembly of Reissner’s fiber. The adrenergic signals activate the expression of Urotensin neuropeptides in the CSF-cNs (Urp1 and Urp2). Finally, the urotensin neuropeptides may further activate the Uts2r3 receptor localized to the dorsal muscle fibers and promote body straightening. Abnormalities in the Urotensin signaling pathway will induce either body curvature in larvae or scoliosis in adult zebrafish
Scoliosis is a common spinal deformity that considerably affects the physical and psychological health of patients. Studies have shown that genetic factors play an important role in scoliosis. However, its etiopathogenesis remain unclear, partially because of the genetic heterogeneity of scoliosis and the lack of appropriate model systems. Recently, the development of efficient gene editing methods and high-throughput sequencing technology has made it possible to explore the underlying pathological mechanisms of scoliosis. Owing to their susceptibility for developing scoliosis and high genetic homology with human, zebrafish are increasingly being used as a model for scoliosis in developmental biology, genetics, and clinical medicine. Here, we summarize the recent advances in scoliosis research on zebrafish and discuss the prospects of using zebrafish as a scoliosis model.
 
Patients with autism spectrum disorder (ASD) typically experience substantial social isolation, which may cause secondary adverse effects on their brain development. miR-124 is the most abundant miRNA in the human brain, acting as a pivotal molecule regulating neuronal fate determination. Alterations of miR-124 maturation or expression are observed in various neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. In the present study, we analyzed a panel of brain-enriched microRNAs in serums from 2 to 6 year old boys diagnosed with ASD. The hsa-miR-124 level was found significantly elevated in ASD boys than in age and sex-matched healthy controls. In an isolation-reared weanling mouse model, we evidenced elevated mmu-miR-124 level in the serum and the medial prefrontal cortex (mPFC). These mice displayed significant sociability deficits, as well as myelin abnormality in the mPFC, which was partially rescued by expressing the miR-124 sponge in the bilateral mPFC, ubiquitously or specifically in oligodendroglia. In cultured mouse oligodendrocyte precursor cells, introducing a synthetic mmu-miR-124 inhibited the differentiation process through suppressing expression of nuclear receptor subfamily 4 group A member 1 (Nr4a1). Overexpressing Nr4a1 in the bilateral mPFC also corrected the social behavioral deficits and myelin impairments in the isolation-reared mice. This study revealed an unanticipated role of the miR-124/Nr4a1 signaling in regulating early social experience-dependent mPFC myelination, which may serve as a potential therapy target for social neglect or social isolation-related neuropsychiatric disorders.
 
In multiple cancers, autophagy promotes tumor development by recycling intracellular components into metabolic pathways. Autophagy-induced metabolic reprogramming and plasticity lead to cancer cell survival and resistance to anticancer therapy. We investigated the role of small leucine zipper protein (sLZIP) in autophagy and cell survival under nutrient-deficient conditions in colorectal cancer (CRC). sLZIP was induced by nutrient stress and increased the transcription of microtubule-associated protein 1A/1B-light chain 3 (LC3), by directly binding to its promoter. Under nutrient stress conditions, sLZIP activated autophagy and promoted the survival of CRC cells. sLZIP induced metabolic reprogramming of CRC cells, to activate glutaminolysis and the tricarboxylic acid cycle. sLZIP also enhanced the autophagic degradation of Keap1 and the nuclear accumulation of Nrf2, leading to NQO1 expression, for maintenance of redox homeostasis. sLZIP-knockout CRC cells exhibited impaired autophagy induction in the glycolytic inhibition state. Xenograft mice lacking sLZIP showed decreased tumor growth, by rendering CRC cells sensitive to glycolysis inhibition. The expression of sLZIP and LC3B was highly elevated in tumors of CRC patients compared to that in normal tissues, and correlated with the progression of CRC. These findings suggest that sLZIP drives autophagy and metabolic reprogramming to promote colorectal tumorigenesis.
 
Background RAS-to-ERK signaling is crucial for the onset and progression of advanced thyroid carcinoma, and blocking ERK dimerization provides a therapeutic benefit in several human carcinomas. Here we analyzed the effects of DEL-22379, a relatively specific ERK dimerization inhibitor, on the activation of the RAS-to-ERK signaling cascade and on tumor-related processes in vitro and in vivo. Methods We used a panel of four human anaplastic thyroid carcinoma (ATC) cell lines harboring BRAF or RAS mutations to analyze ERK dynamics and tumor-specific characteristics. We also assessed the impact of DEL-22379 on the transcriptional landscape of ATC cell lines using RNA-sequencing and evaluated its therapeutic efficacy in an orthotopic mouse model of ATC. Results DEL-22379 impaired upstream ERK activation in BRAF- but not RAS-mutant cells. Cell viability and metastasis-related processes were attenuated by DEL-22379 treatment, but mostly in BRAF-mutant cells, whereas in vivo tumor growth and dissemination were strongly reduced for BRAF-mutant cells and mildly reduced for RAS-mutant cells. Transcriptomics analyses indicated that DEL-22379 modulated the transcriptional landscape of BRAF- and RAS-mutant cells in opposite directions. Conclusions Our findings establish that BRAF- and RAS-mutant thyroid cells respond differentially to DEL-22379, which cannot be explained by the previously described mechanism of action of the inhibitor. Nonetheless, DEL-22379 demonstrated significant anti-tumor effects against BRAF-mutant cells in vivo with an apparent lack of toxicity, making it an interesting candidate for the development of combinatorial treatments. Our data underscore the differences elicited by the specific driver mutation for thyroid cancer onset and progression, which should be considered for experimental and clinical approaches.
 
Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients’ respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.
 
Illustration for the study design of the animal experiments. a Illustration for the study design of the first animal experiment. This experiment included four groups (n = 4 per group) of mice. The first group served as the control group. The second group are wild type FVB-NJ mice in which colorectal cancer (CRC) was chemically induced. The third group composed of MKR diabetic mice. The fourth group included MKR diabetic mice in which CRC was chemically induced. CRC was induced by intraperitoneally injecting the mice with the genotoxin, Azoxymethane (AOM), (10 mg/kg) then after one week, mice were supplied with dextran sulfate sdium (DSS), 2.5% solution instead of drinking water for one week. Mice were allowed to rest for 2 weeks then the DSS cycle was repeated for 3 more times. Fecal samples were collected at an early time point (age: 7 weeks) and a late time point (age: 31 weeks). b Illustration for the study design of the second animal experiment. In this experiment, FVB-NJ mice were depleted from their endogenous microbiota by using an antibiotic cocktail after weaning (age: 3 weeks). The antibiotic cocktail was supplied in the drinking water of mice on alternative days for 5 weeks. After the depletion of microbiota, FVB-NJ mice were divided into five groups. The first group was left without any microbial transplant. The second group was inoculated with fecal microbial transplant (FMT) from FVB-NJ mice from the first experiment. The third group was inoculated with FMT from “FVB CRC” mice from the first experiment. The fourth group was inoculated with FMT from MKR diabetic mice from the first experiment. The fifth group was inoculated with FMT from “MKR CRC” mice from the first experiment. After the FMT, colorectal cancer was chemically induced in all mice groups. CRC was induced by intraperitoneally injecting the mice with the genotoxin, Azoxymethane (AOM), (10 mg/kg) then after one week, mice were supplied with dextran sulfate sodium (DSS), 2.5% solution instead of drinking water for one week. Mice were allowed to rest for 2 weeks then the DSS cycle was repeated for 3 more times. Fecal samples were collected at an early time point (age: 7 weeks) and a late time point (age: 31 weeks). c Illustration for the study design of the third animal experiment. The first group included FVB-NJ mice that served as the control group. The second group included FVB-NJ mice in which colorectal cancer (CRC) was induced followed by treatment with vehicle (Phosphate-buffered saline “PBS”). The third group included FVB-NJ mice in which colorectal cancer (CRC) was induced followed by treatment with Probiotics formula (ProBioLife, 5 mg/kg once daily by oral gavage for 12 weeks). The fourth group included MKR diabetic mice treated with vehicle (Phosphate-buffered saline “PBS”). The fifth group included MKR diabetic mice treated with Probiotics formula (ProBioLife, 5 mg/kg once daily by oral gavage for 12 weeks). Treatment started at week 19 of age and ended at week 31. CRC colorectal cancer, AOM Azoxymethane, DSS Dextran sulfate sodium, FMT Fecal microbial transplant
Colorectal cancer polyps in different mice groups. a Gross characteristics for colorectal cancer (CRC) and a scatter plot representing the number of polyps in FVB-NJ CRC and MKR CRC mice groups. Values are means ± SEM of 3 animals per group (n = 3/group). *p < 0.05 vs. FVB CRC mice. b A scatter plot representing the polyp numbers of mice inoculated with fecal microbial transplant and representative images of the polyps. Values are means ± SEM of 3 animals per group (n = 3/group). $p < 0.05 vs. No microbiota and #p < 0.05 vs. FVB-FMT group. Results are expressed as mean ± SD. c Representative images of hematoxylin and eosin (H&E)‐stained colon sections illustrating the histological changes in FVB control, FVB CRC, CRC probiotics, MKR diabetic, MKR CRC and MKR probiotics groups. The worst alterations were encountered in the MKR CRC group. Note the presence of adenomatous development (square), hyperplasia(circle), inflammatory cells invading the edematous submucosa (star), crypt abscess (black triangle), as well as crypt architecture disarray (arrow)
Microbiota composition as revealed by 16S rRNA sequencing. a A box plot representing alpha diversity in FVB control, MKR diabetic, FVB with colorectal cancer (CRC) and MKR CRC mice at early and late time points (n = 3/group). b The principal coordinate analysis (PCoA) plot for FVB control, MKR diabetic, FVB with colorectal cancer (CRC) and MKR CRC mice at early and late time points (n = 3/group). c A bar graph representing the relative abundance of each bacterial phyla in different samples obtained from FVB control, MKR diabetic, FVB with colorectal cancer (CRC) and MKR CRC mice at early and late time points (n = 3/group). d A box plot representing alpha diversity in mice with no microbiota, FVB FMT (fecal microbial transplant), MKR FMT, FVB CRC FMT and MKR CRC FMT at early and late time points (n = 3/group). e Rarefaction curve of samples obtained from mice with no microbiota, FVB FMT, MKR FMT, FVB CRC FMT and MKR CRC FMT at early and late time points (n = 3/group). f A bar graph representing the relative abundance of each bacterial phyla in different samples obtained from mice with no microbiota, FVB FMT, MKR FMT, FVB CRC FMT and MKR CRC FMT at early and late time points (n = 3/group). g PCoA plot for mice with no microbiota, FVB FMT, MKR FMT, FVB CRC FMT and MKR CRC FMT at early and late time points (n > 2/group). CRC colorectal cancer, FMT fecal microbial transplant, PCoA principal coordinate analysis
Pie charts of microbiota composition of different mice groups at early and late time points showing mean relative abundance of major bacterial phyla as well as the fraction of the butyrate-producing bacteria
Protein expression of interleukin (IL)-1β and NADPH oxidase (NOX)4 in different mice groups. a A scatter plot representing the quantification of protein expression of IL-1β in colon tissues of FVB control, MKR diabetic, FVB with colorectal cancer (CRC) and MKR CRC mice. Values are means ± SEM of 3 animals per group (n = 3/group). *Statistically significant at p < 0.05 vs FVB control group. b A scatter plot representing the quantification of protein expression of NOX4 in colon tissues of FVB control, MKR diabetic, FVB with colorectal cancer (CRC) and MKR CRC mice. Values are means ± SEM of 3 animals per group (n = 3/group). *Statistically significant at p < 0.05 vs FVB control group. c a scatter plot representing the quantification of protein expression of IL-1β in colon tissues of for mice with no microbiota, FVB FMT(fecal microbial transplant), MKR FMT, FVB CRC FMT and MKR CRC FMT. Values are means ± SEM of 3 animals per group (n = 3/group). d A scatter plot representing the quantification of protein expression of NOX4 in colon tissues of for mice with no microbiota, FVB FMT, MKR FMT, FVB CRC FMT and MKR CRC FMT. Values are means ± SEM of 3 animals per group (n = 3/group). e Colon lengths of FVB control, MKR diabetic treated with vehicle (PBS), FVB CRC treated with vehicle (PBS), MKR diabetic treated with probiotics for 12 weeks and FVB CRC treated with probiotics for 12 weeks. Values are means ± SEM of 3 animals per group (n = 3/group). *Statistically significant at p < 0.05 vs FVB control group. #Statistically significant at p < 0.05 vs MKR Diabetic. $ statistically significant at p < 0.05 vs FVB CRC. f A scatter plot representing the polyp numbers of FVB CRC mice treated with vehicle (PBS) or probiotics for 12 weeks and representative images of the polyps. Values are means ± SEM of 3 animals per group (n = 3/group). $Statistically significant at p < 0.05 vs FVB CRC. g A scatter plot representing the quantification of protein expression of IL-1β in colon tissues of FVB control, MKR diabetic, and MKR mice treated with probiotics for 12 weeks. Values are means ± SEM of 3 animals per group (n = 3/group). *Statistically significant at p < 0.05 vs FVB control group. #Statistically significant at p < 0.05 vs MKR Diabetic. h A scatter plot representing the quantification of protein expression of IL-1β in colon tissues of FVB control, FVB CRC mice, FVB CRC mice treated with probiotics for 12 weeks. Values are means ± SEM of 3 animals per group (n = 3/group). *Statistically significant at p < 0.05 vs FVB control group. #Statistically significant at p < 0.05 vs MKR Diabetic. i A scatter plot representing the quantification of protein expression of NOX4 in colon tissues of FVB control, MKR diabetic, and MKR mice treated with probiotics for 12 weeks. Values are means ± SEM of 3 animals per group (n = 3/group). *Statistically significant at p < 0.05 vs FVB control group. # statistically significant at p < 0.05 vs MKR Diabetic. j A scatter plot representing the quantification of protein expression of NOX4 in colon tissues of FVB control, FVB CRC mice, FVB CRC mice treated with probiotics for 12 weeks. Values are means ± SEM of 3 animals per group (n = 3/group). *Statistically significant at p < 0.05 vs FVB control group. #Statistically significant at p < 0.05 vs MKR Diabetic. CRC Colorectal cancer, FMT Fecal microbial transplant, IL interleukin, NOX NADPH oxidase, PBS phosphate-buffered saline
Diabetes changes the host microbiota, a condition known as dysbiosis. Dysbiosis is an important factor for the pathogenesis of diabetes and colorectal cancer (CRC). We aimed at identifying the microbial signature associated with diabetes and CRC; and identifying the signaling mechanism altered by dysbiosis and leading to CRC progression in diabetes. MKR mice that can spontaneously develop type 2 diabetes were used. For CRC induction, another subset of mice was treated with azoxymethane and dextran sulfate sodium. To identify the role of microbiota, microbiota-depleted mice were inoculated with fecal microbial transplant from diabetic and CRC mice. Further, a mouse group was treated with probiotics. At the end of the treatment, 16S rRNA sequencing was performed to identify microbiota in the fecal samples. Blood was collected, and colons were harvested for molecular, anatomical, and histological analysis. Our results show that diabetes is associated with a microbial signature characterized by reduction of butyrate-forming bacteria. This dysbiosis is associated with gastrointestinal complications reflected by a reduction in colon lengths. These changes are reversed upon treatment with probiotics, which rectified the observed dysbiosis. Inoculation of control mice with diabetic or cancer microbiota resulted in the development of increased number of polyps. Our data also show that inflammatory cytokines (mainly interleukin (IL)-1β) and NADPH oxidase (NOX)4 are over-expressed in the colon tissues of diabetic mice. Collectively our data suggest that diabetes is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria leading to over-expression of IL-1β and NOX4 leading to gastrointestinal complications and CRC.
 
Background Poly-GA, a dipeptide repeat protein unconventionally translated from GGGGCC (G4C2) repeat expansions in C9orf72, is abundant in C9orf72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9orf72-ALS/FTD). Although the poly-GA aggregates have been identified in C9orf72-ALS/FTD neurons, the effects on UPS (ubiquitin–proteasome system) and autophagy and their exact molecular mechanisms have not been fully elucidated.ResultsHerein, our in vivo experiments indicate that the mice expressing ploy-GA with 150 repeats instead of 30 repeats exhibit significant aggregates in cells. Mice expressing 150 repeats ploy-GA shows behavioral deficits and activates autophagy in the brain. In vitro findings suggest that the poly-GA aggregates influence proteasomal by directly binding proteasome subunit PSMD2. Subsequently, the poly-GA aggregates activate phosphorylation and ubiquitination of p62 to recruit autophagosomes. Ultimately, the poly-GA aggregates lead to compensatory activation of autophagy. In vivo studies further reveal that rapamycin (autophagy activator) treatment significantly improves the degenerative symptoms and alleviates neuronal injury in mice expressing 150 repeats poly-GA. Meanwhile, rapamycin administration to mice expressing 150 repeats poly-GA reduces neuroinflammation and aggregates in the brain.Conclusion In summary, we elucidate the relationship between poly-GA in the proteasome and autophagy: when poly-GA forms complexes with the proteasome, it recruits autophagosomes and affects proteasome function. Our study provides support for further promoting the comprehension of the pathogenesis of C9orf72, which may bring a hint for the exploration of rapamycin for the treatment of ALS/FTD.
 
Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca ²⁺ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca ²⁺ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ 42 -derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca ²⁺ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca ²⁺ than ROS levels, and a lag phase in the latter process. A Ca ²⁺ -deprived cell medium prevented the increase of intracellular Ca ²⁺ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca ²⁺ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca ²⁺ causes the production of ROS in cells challenged by aberrant protein oligomers.
 
Predicted spatial protein structure of human APOE (AlphaFold) and intracellular localisation of APOE. a The APOE isoproteins differ at one and two amino acid (AA) positions, respectively (112 and 158). This affects the structure and thus the function of the protein, e.g., APOE2 shows a significantly reduced affinity for the low-density lipoprotein receptor (LDLR) and the lipid binding preference differs between the isoforms. The different colours of the protein structure indicate the model confidence of the structure prediction (dark blue, very high; light blue, confident; yellow, low; orange, very low). Protein structure image downloaded from https://alphafold.ebi.ac.uk/. HDL high-density lipoprotein, VLDL very low-density lipoprotein. b Schematic illustration of an eukaryotic cell and the individual cell organelles and compartments. APOE is known to be a secreted protein, but has been detected inside the cell in, e.g., mitochondria, peroxisomes, and the nucleus. MAMs mitochondria-associated ER membranes
Illustration of the assembly of mitochondria and mitochondria–ER contacts and the inherent biochemical pathways with suggested APOE involvement or regulation by the APOE isoform. a Mitochondria-associated ER membrane complex (MAM). b Mitochondrial and MAM pathways in which APOE may be involved. (I) The impact of APOE isoforms on mitochondrial function depends on the cell type and species, but consistently decreased neuronal OXPHOS protein and ATP levels were observed in APOE4. (II) Mitochondrial accumulation and stress-induced translocation are increased in APOE4. (III) The different steps in phospholipid synthesis take place through consecutive exchange of substrates from the ER membrane (synthesis of phosphatidylserine; PS) to the mitochondrion (conversion of PS to phosphatidylethanolamine (PE), which is increased in APOE4) and back. The final methylation step by PEMT is accomplished in the ER membrane, yielding phosphatidylcholine (PC). (IV) Calcium is released from the ER through the IP3R1–GRP75–VDAC1 complex and shuttled to the mitochondrion through the mitochondrial calcium uniporter protein (MCU) into the inner matrix. Increased calcium flux was found in APOE4 Neuro2a cells, and higher mitochondrial swelling was observed in APOE4-treated H9c2 cells, which was caused by the interaction of APOE (derived from the lysosomal degradation of L5-LDL) with VDAC1. The protein–protein interaction of APOE with MAM proteins such as VDAC1 and GRP75 is one explanation for the presence of APOE in MAMs. (V) MFN2 dimers connect the OMM with the ER membrane, acting as MAM tethering proteins. Depending on the tissue and species, APOE affects the expression of MFN1 and MFN2. (VI) MAMs are involved in cholesterol metabolism, and proteomic analyses provide evidence for a possible role of APOE in VLDL assembly in MAMs
Potential APOE protein–protein interactions in human cells and tissue. An in silico analysis performed with BioGRID 4.4 software revealed that there is evidence for 145 potential protein binding partners whose physical interaction with APOE has been demonstrated in at least one study in each case. The larger the blue circle of the corresponding protein is, the stronger the connectivity with APOE, and thicker binding lines represent stronger evidence supporting the association. Image downloaded from https://thebiogrid.org/
Human apolipoprotein E (APOE), originally known for its role in lipid metabolism, is polymorphic with three major allele forms, namely, APOEε2, APOEε3, and APOEε4, leading to three different human APOE isoforms. The ε4 allele is a genetic risk factor for Alzheimer’s disease (AD); therefore, the vast majority of APOE research focuses on its role in AD pathology. However, there is increasing evidence for other functions of APOE through the involvement in other biological processes such as transcriptional regulation, mitochondrial metabolism, immune response, and responsiveness to dietary factors. Therefore, the aim of this review is to provide an overview of the potential novel functions of APOE and their characterization. The detection of APOE in various cell organelles points to previously unrecognized roles in mitochondria and others, although it is actually considered a secretory protein. Furthermore, numerous interactions of APOE with other proteins have been detected, providing indications for new metabolic pathways involving APOE. The present review summarizes the current evidence on APOE beyond its original role in lipid metabolism, to change the perspective and encourage novel approaches to future research on APOE and its isoform-dependent role in the cellular metabolism.
 
Sertoli cells contribute to the formation of the blood-testis barrier (BTB), which is necessary for normal spermatogenesis. Recently, microRNAs (miRNAs) have emerged as posttranscriptional regulatory elements in BTB function during spermatogenesis. Our previous study has shown that miR-181c or miR-181d (miR-181c/d) is highly expressed in testes from boars at 60 days old compared with at 180 days old. Herein, we found that overexpression of miR-181c/d via miR-181c/d mimics in murine Sertoli cells (SCs) or through injecting miR-181c/d-overexpressing lentivirus in murine testes perturbs BTB function by altering BTB-associated protein distribution at the Sertoli cell–cell interface and F-actin organization, but this in vivo perturbation disappears approximately 6 weeks after the final treatment. We also found that miR-181c/d represses Sertoli cell proliferation and promotes its apoptosis. Moreover, miR-181c/d regulates Sertoli cell survival and barrier function by targeting platelet-activating factor acetylhydrolase 1b regulatory subunit 1 ( Pafah1b1 ) gene. Furthermore, miR-181c/d suppresses PAFAH1B1 expression, reduces the complex of PAFAH1B1 with IQ motif-containing GTPase activating protein 1, and inhibits CDC42/PAK1/LIMK1/Cofilin pathway which is required for F-actin stabilization. In total, our results reveal the regulatory axis of miR-181c/d- Pafah1b1 in cell survival and barrier function of Sertoli cells and provide additional insights into miRNA functions in mammalian spermatogenesis.
 
Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.
 
The PTM-landscape of SMN. The SMN protein is encoded by 8 exons comprising different conserved domains. The depicted PTM sites are identified by mass spectrometry/proteomics and other methods (see also Table 1). P phosphorylation, Ac acetylation, Ub/UbUbUb mono-/polyubiquitinylation, M methylation
Altered phosphorylation of the SMN protein in SMA patients. To date, there are 26 phosphorylation sites within the SMN protein sequence, which have been identified by mass spectrometry. To our knowledge, 12 point mutations of SMN found in SMA patients are putative phosphorylation sites. Only 5 of these known SMA patient mutations are also identified as phosphorylation sites. The residual 7 point mutations remain putative phosphorylation sites within the SMN protein
Conservation of MS-identified phosphorylation sites of the SMN protein. The multiple alignment of the SMN protein sequences of 24 species including fungi, arthropods, nematodes, fish, birds, reptiles, amphibians and mammals was performed with the freeware CLUSTALW [79]. The conservation of the phosphorylation sites within the 8 encoding exons was further analyzed between the 24 species. The conservation of a phospho-site found in 24 of 24 species (24/24) was set as 100%. For a translational perspective the conservation of SMN phosphorylation sites between human and mouse was additionally determined
Spinal muscular atrophy (SMA) is caused by low levels of the survival of motoneuron (SMN) Protein leading to preferential degeneration of lower motoneurons in the ventral horn of the spinal cord and brain stem. However, the SMN protein is ubiquitously expressed and there is growing evidence of a multisystem phenotype in SMA. Since a loss of SMN function is critical, it is important to decipher the regulatory mechanisms of SMN function starting on the level of the SMN protein itself. Posttranslational modifications (PTMs) of proteins regulate multiple functions and processes, including activity, cellular trafficking, and stability. Several PTM sites have been identified within the SMN sequence. Here, we map the identified SMN PTMs highlighting phosphorylation as a key regulator affecting localization, stability and functions of SMN. Furthermore, we propose SMN phosphorylation as a crucial factor for intracellular interaction and cellular distribution of SMN. We outline the relevance of phosphorylation of the spinal muscular atrophy (SMA) gene product SMN with regard to basic housekeeping functions of SMN impaired in this neurodegenerative disease. Finally, we compare SMA patient mutations with putative and verified phosphorylation sites. Thus, we emphasize the importance of phosphorylation as a cellular modulator in a clinical perspective as a potential additional target for combinatorial SMA treatment strategies.
 
Loss of cyclin-dependent kinase 5 (Cdk5) in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) increases ER–mitochondria tethering and ER Ca ²⁺ transfer to the mitochondria, subsequently increasing mitochondrial Ca ²⁺ concentration ([Ca ²⁺ ] mt ). This suggests a role for Cdk5 in regulating intracellular Ca ²⁺ dynamics, but how Cdk5 is involved in this process remains to be explored. Using ex vivo primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5 −/− mouse embryos, we show here that loss of Cdk5 causes an increase in cytosolic Ca ²⁺ concentration ([Ca ²⁺ ] cyt ), which is not due to reduced internal Ca ²⁺ store capacity or increased Ca ²⁺ influx from the extracellular milieu. Instead, by stimulation with ATP that mediates release of Ca ²⁺ from internal stores, we determined that the rise in [Ca ²⁺ ] cyt in Cdk5 −/− MEFs is due to increased inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca ²⁺ release from internal stores. Cdk5 interacts with the IP3R1 Ca ²⁺ channel and phosphorylates it at Ser 421 . Such phosphorylation controls IP3R1-mediated Ca ²⁺ release as loss of Cdk5, and thus, loss of IP3R1 Ser 421 phosphorylation triggers an increase in IP3R1-mediated Ca ²⁺ release in Cdk5 −/− MEFs, resulting in elevated [Ca ²⁺ ] cyt . Elevated [Ca ²⁺ ] cyt in these cells further induces the production of reactive oxygen species (ROS), which upregulates the levels of Nrf2 and its targets, Prx1 and Prx2. Cdk5 −/− MEFs, which have elevated [Ca ²⁺ ] cyt , proliferate at a faster rate compared to wt, and Cdk5 −/− embryos have increased body weight and size compared to their wt littermates. Taken together, we show that altered IP3R1-mediated Ca ²⁺ dynamics due to Cdk5 loss correspond to accelerated cell proliferation that correlates with increased body weight and size in Cdk5 −/− embryos.