Generation of embryo-like structures from mouse embryonic stem cells treated with a chemical inhibitor of SUMOylation and cultured in microdroplets.

The field of stem cell-based embryo-like models is rapidly evolving, providing in vitro models of in utero stages of mammalian development. Here, we detail steps to first establish adherent spheroids composed of three cell types from mouse embryonic stem cells solely treated with a chemical inhibitor of SUMOylation. We then describe procedures for generating highly reproducible gastruloids from these dissociated spheroid cells, as well as embryo-like structures comprising anterior neural and trunk somite-like regions using an optimized microfluidics platform. For complete details on the use and execution of this protocol, please refer to Cossec et al. (2023).1.

Transient suppression of SUMOylation in embryonic stem cells generates embryo-like structures.

Recent advances in synthetic embryology have opened new avenues for understanding the complex events controlling mammalian peri-implantation development. Here, we show that mouse embryonic stem cells (ESCs) solely exposed to chemical inhibition of SUMOylation generate embryo-like structures comprising anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs give rise to spheroids that self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in a droplet microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell transcriptomics reveals various cellular lineages, including properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures. Transient SUMOylation suppression gradually increases DNA methylation genome wide and repressive mark deposition at Nanog. Interestingly, cell-to-cell variations in SUMOylation levels occur during early embryogenesis. Our approach provides a proof of principle for potentially powerful strategies to explore early embryogenesis by targeting chromatin roadblocks of cell fate change.

Extensive SUMO Modification of Repressive Chromatin Factors Distinguishes Pluripotent from Somatic Cells.

Post-translational modification by SUMO is a key regulator of cell identity. In mouse embryonic fibroblasts (MEFs), SUMO impedes reprogramming to pluripotency, while in embryonic stem cells (ESCs), it represses the emergence of totipotent-like cells, suggesting that SUMO targets distinct substrates to preserve somatic and pluripotent states. Using MS-based proteomics, we show that the composition of endogenous SUMOylomes differs dramatically between MEFs and ESCs. In MEFs, SUMO2/3 targets proteins associated with canonical SUMO functions, such as splicing, and transcriptional regulators driving somatic enhancer selection. In contrast, in ESCs, SUMO2/3 primarily modifies highly interconnected repressive chromatin complexes, thereby preventing chromatin opening and transitioning to totipotent-like states. We also characterize several SUMO-modified pluripotency factors and show that SUMOylation of Dppa2 and Dppa4 impedes the conversion to 2-cell-embryo-like states. Altogether, we propose that rewiring the repertoire of SUMO target networks is a major driver of cell fate decision during embryonic development.

Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome.

Enlarged early endosomes have been visualized in Alzheimer's disease (AD) and Down syndrome (DS) using conventional confocal microscopy at a resolution corresponding to endosomal size (hundreds of nm). In order to overtake the diffraction limit, we used super-resolution structured illumination microscopy (SR-SIM) and transmission electron microscopies (TEM) to analyze the early endosomal compartment in DS.By immunofluorescence and confocal microscopy, we confirmed that the volume of Early Endosome Antigen 1 (EEA1)-positive puncta was 13-19% larger in fibroblasts and iPSC-derived neurons from individuals with DS, and in basal forebrain cholinergic neurons (BFCN) of the Ts65Dn mice modelling DS. However, EEA1-positive structures imaged by TEM or SR-SIM after chemical fixation had a normal size but appeared clustered. In order to disentangle these discrepancies, we imaged optimally preserved High Pressure Freezing (HPF)-vitrified DS fibroblasts by TEM and found that early endosomes were 75% denser but remained normal-sized.RNA sequencing of DS and euploid fibroblasts revealed a subgroup of differentially-expressed genes related to cargo sorting at multivesicular bodies (MVBs). We thus studied the dynamics of endocytosis, recycling and MVB-dependent degradation in DS fibroblasts. We found no change in endocytosis, increased recycling and delayed degradation, suggesting a "traffic jam" in the endosomal compartment.Finally, we show that the phosphoinositide PI (3) P, involved in early endosome fusion, is decreased in DS fibroblasts, unveiling a new mechanism for endosomal dysfunctions in DS and a target for pharmacotherapy.

SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States.

Understanding general principles that safeguard cellular identity should reveal critical insights into common mechanisms underlying specification of varied cell types. Here, we show that SUMO modification acts to stabilize cell fate in a variety of contexts. Hyposumoylation enhances pluripotency reprogramming in vitro and in vivo, increases lineage transdifferentiation, and facilitates leukemic cell differentiation. Suppressing sumoylation in embryonic stem cells (ESCs) promotes their conversion into 2-cell-embryo-like (2C-like) cells. During reprogramming to pluripotency, SUMO functions on fibroblastic enhancers to retain somatic transcription factors together with Oct4, Sox2, and Klf4, thus impeding somatic enhancer inactivation. In contrast, in ESCs, SUMO functions on heterochromatin to silence the 2C program, maintaining both proper H3K9me3 levels genome-wide and repression of the Dux locus by triggering recruitment of the sumoylated PRC1.6 and Kap/Setdb1 repressive complexes. Together, these studies show that SUMO acts on chromatin as a glue to stabilize key determinants of somatic and pluripotent states.

Sumoylation coordinates the repression of inflammatory and anti-viral gene-expression programs during innate sensing.

Innate sensing of pathogens initiates inflammatory cytokine responses that need to be tightly controlled. We found here that after engagement of Toll-like receptors (TLRs) in myeloid cells, deficient sumoylation caused increased secretion of transcription factor NF-κB-dependent inflammatory cytokines and a massive type I interferon signature. In mice, diminished sumoylation conferred susceptibility to endotoxin shock and resistance to viral infection. Overproduction of several NF-κB-dependent inflammatory cytokines required expression of the type I interferon receptor, which identified type I interferon as a central sumoylation-controlled hub for inflammation. Mechanistically, the small ubiquitin-like modifier SUMO operated from a distal enhancer of the gene encoding interferon-ß (Ifnb1) to silence both basal and stimulus-induced activity of the Ifnb1 promoter. Therefore, sumoylation restrained inflammation by silencing Ifnb1 expression and by strictly suppressing an unanticipated priming by type I interferons of the TLR-induced production of inflammatory cytokines.

Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation.

Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.

Homodimerization of amyloid precursor protein at the plasma membrane: a homoFRET study by time-resolved fluorescence anisotropy imaging.

Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer's disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions.

Trisomy for synaptojanin1 in Down syndrome is functionally linked to the enlargement of early endosomes.

Enlarged early endosomes have been observed in neurons and fibroblasts in Down syndrome (DS). These endosome abnormalities have been implicated in the early development of Alzheimer's disease (AD) pathology in these subjects. Here, we show the presence of enlarged endosomes in blood mononuclear cells and lymphoblastoid cell lines (LCLs) from individuals with DS using immunofluorescence and confocal microscopy. Genotype-phenotype correlations in LCLs carrying partial trisomies 21 revealed that triplication of a 2.56 Mb locus in 21q22.11 is associated with the endosomal abnormalities. This locus contains the gene encoding the phosphoinositide phosphatase synaptojanin 1 (SYNJ1), a key regulator of the signalling phospholipid phosphatidylinositol-4,5-biphosphate that has been shown to regulate clathrin-mediated endocytosis. We found that SYNJ1 transcripts are increased in LCLs from individuals with DS and that overexpression of SYNJ1 in a neuroblastoma cell line as well as in transgenic mice leads to enlarged endosomes. Moreover, the proportion of enlarged endosomes in fibroblasts from an individual with DS was reduced after silencing SYNJ1 expression with RNA interference. In LCLs carrying amyloid precursor protein (APP) microduplications causing autosomal dominant early-onset AD, enlarged endosomes were absent, suggesting that APP overexpression alone is not involved in the modification of early endosomes in this cell type. These findings provide new insights into the contribution of SYNJ1 overexpression to the endosomal changes observed in DS and suggest an attractive new target for rescuing endocytic dysfunction and lipid metabolism in DS and in AD.

Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis.

Amyloid peptide (Aß) is generated by sequential cleavage of the amyloid precursor protein (APP) by ß-secretase (Bace1) and γ-secretase. Aß production increases after plasma membrane cholesterol loading through unknown mechanisms. To determine how APP-Bace1 proximity affects this phenomenon, we developed a fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET) technique for visualization of these molecules either by epifluorescence or at the plasma membrane only using total internal reflection fluorescence. Further, we used fluorescence correlation spectroscopy to determine the lipid rafts partition of APP-yellow fluorescent protein (YFP) and Bace1-green fluorescent protein (GFP) molecules at the plasma membrane of neurons. We show that less than 10 min after cholesterol exposure, Bace1-GFP/APP-mCherry proximity increases selectively at the membrane and APP relocalizes to raft domains, preceded by rapid endocytosis. After longer cholesterol exposures, APP and Bace1 are found in proximity intracellularly. We demonstrate that cholesterol loading does not increase Aß production by having a direct impact on Bace1 catalytic activity but rather by altering the accessibility of Bace1 to its substrate, APP. This change in accessibility is mediated by clustering in lipid rafts, followed by rapid endocytosis.

Clathrin-dependent APP endocytosis and Abeta secretion are highly sensitive to the level of plasma membrane cholesterol.

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-beta (Abeta) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to +40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Abeta.

Cholesterol changes in Alzheimer's disease: methods of analysis and impact on the formation of enlarged endosomes.

An increasing number of results implicating cholesterol metabolism in the pathophysiology of Alzheimer's disease (AD) suggest cholesterol as a target for treatment. Research in genetics, pathology, epidemiology, biochemistry, and cell biology, as well as in animal models, suggests that cholesterol, its transporter in the brain, apolipoprotein E, amyloid precursor protein, and amyloid-beta all interact in AD pathogenesis. Surprisingly, key questions remain unanswered due to the lack of sensitive and specific methods for assessing cholesterol levels in the brain at subcellular resolution. The aims of this review are not only to discuss the various methods for measuring cholesterol and its metabolites and to catalog results obtained from AD patients but also to discuss some new data linking high plasma membrane cholesterol with modifications of the endocytic compartments. These studies are particularly relevant to AD pathology, since enlarged endosomes are believed to be the first morphological change observed in AD brains, in both sporadic cases and Down syndrome.

Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry.

Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimer's disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimer's disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimer's disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-beta peptide (A beta) deposit. Two cases were apo epsilon 4/apo epsilon 3, and one case was apo epsilon 3/apoepsilon3. A known quantity of (13)C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 +/- 0.1 attomoles/microm(3) in the senile plaques and 2.2 +/- 0.49 attomoles/microm(3) in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 +/- 16 femtomol) is similar to the published quantity of A beta peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimer's disease linked to the apo epsilon 4 allele, suggests new pathogenetic mechanisms.

Mutations in GLIS3 are responsible for a rare syndrome with neonatal diabetes mellitus and congenital hypothyroidism.

We recently described a new neonatal diabetes syndrome associated with congenital hypothyroidism, congenital glaucoma, hepatic fibrosis and polycystic kidneys. Here, we show that this syndrome results from mutations in GLIS3, encoding GLI similar 3, a recently identified transcription factor. In the original family, we identified a frameshift mutation predicted to result in a truncated protein. In two other families with an incomplete syndrome, we found that affected individuals harbor deletions affecting the 11 or 12 5'-most exons of the gene. The absence of a major transcript in the pancreas and thyroid (deletions from both families) and an eye-specific transcript (deletion from one family), together with residual expression of some GLIS3 transcripts, seems to explain the incomplete clinical manifestations in these individuals. GLIS3 is expressed in the pancreas from early developmental stages, with greater expression in beta cells than in other pancreatic tissues. These results demonstrate a major role for GLIS3 in the development of pancreatic beta cells and the thyroid, eye, liver and kidney.