Modulation of Unfolded Protein Response Restores Survival and Function of β-Cells Exposed to the Endocrine Disruptor Bisphenol A

Diabetes is a metabolic disease that currently affects nearly half a billion people worldwide. β-cells dysfunction is one of the main causes of diabetes. Exposure to endocrine-disrupting chemicals is correlated with increased diabetes incidence. We hypothesized that treatment with bisphenol A (BPA) induces endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR), leading to impaired function of the β-cells, which over time, can cause diabetes. In this study, we aimed to evaluate UPR pathways activation under BPA treatment in β-cells and possible recovery of ER homeostasis. MIN6 cells (mouse insulinoma cell line) and isolated pancreatic islets from NOR (non-obese diabetes resistant) mice were treated with BPA. We analyzed the impact of BPA on β-cell viability, the architecture of the early secretory pathway, the synthesis and processing of insulin and the activation of UPR sensors and effectors. We found that the addition of the chemical chaperone TUDCA rescues the deleterious effects of BPA, resulting in improved viability, morphology and function of the β-cells. In conclusion, we propose that modulators of UPR can be used as therapeutic interventions targeted towards regaining β-cells homeostasis.publishedVersio

Reprogrammed Cells Display Distinct Proteomic SignaturesAssociated with Colony Morphology Variability

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity.publishedVersio

The Effect of Wnt Pathway Modulators on Human iPSC-Derived Pancreatic Beta Cell Maturation

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation

Analysis of LNP-1 involvement in synaptic vesicle trafficking and neurotransmission

Récemment, un nouveau gène de souris, Inp, a été identifié en amont de cluster HoxD. Chez C. elegans, Inp-1 code pour une protéine putative qui contient les domaines de séquences conservés parmi tous les orthologues actuellement identifiés. Inp-1 est principalement exprimé dans les cellules neuronales chez le ver et colocalise avec les marqueurs de vésicules synaptiques dans les cultures cellulaires de mammifères. Les animaux déficients pour Inp-1 présentent une augmentation significative de leur résistance à l'aldicarb comparé aux animaux de type sauvage. Encore, la mauvaise localisation des protéines presynaptiques, telles que synaptobrevin-1 ou RAB-3 chez les mutants Inp-1, suggère une fonction pour LNP-1 dans la neutrotransmission. Nous avons aussi identifié un partenaire putatif de LNP-1: SIAH-1, une ligase E3-ubiquitin. Elucider les mécanismes moléculaires par lesquels le système LNP-1-ubiquitine-protéasome régule la neurotransmission pourrait être un aspect essentiel pour comprendre les processus pathologiques qui sous-tendent certains des désordres du système nerveux central

Cell plasticity in homeostasis and regeneration

Over the past decades, genetic analyses performed in vertebrate and invertebrate organisms deciphered numerous cellular and molecular mechanisms deployed during sexual development and identified genetic circuitries largely shared among bilaterians. In contrast, the functional analysis of the mechanisms that support regenerative processes in species randomly scattered among the animal kingdom, were limited by the lack of genetic tools. Consequently, unifying principles explaining how stress and injury can lead to the reactivation of a complete developmental program with restoration of original shape and function remained beyond reach of understanding. Recent data on cell plasticity suggest that beside the classical developmental approach, the analysis of homeostasis and asexual reproduction in adult organisms provides novel entry points to dissect the regenerative potential of a given species, a given organ or a given tissue. As a clue, both tissue homeostasis and regeneration dynamics rely on the availability of stem cells and/or on the plasticity of differentiated cells to replenish the missing structure. The freshwater Hydra polyp provides us with a unique model system to study the intricate relationships between the mechanisms that regulate the maintenance of homeostasis, even in extreme conditions (starvation and overfeeding) and the reactivation of developmental programs after bisection or during budding. Interestingly head regeneration in Hydra can follow several routes according to the level of amputation, suggesting that indeed the homeostatic background dramatically influences the route taken to bridge injury and regeneration

Injury-induced activation of the MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in hydra head regeneration

After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process

Autophagy in Hydra: a response to starvation and stress in early animal evolution

The Hydra polyp provides a powerful model system to investigate the regulation of cell survival and cell death in homeostasis and regeneration as Hydra survive weeks without feeding and regenerates any missing part after bisection. Induction of autophagy during starvation is the main surviving strategy in Hydra as autophagic vacuoles form in most myoepithelial cells after several days. When the autophagic process is inhibited, animal survival is actually rapidly jeopardized. An appropriate regulation of autophagy is also essential during regeneration as Hydra RNAi knocked-down for the serine protease inhibitor Kazal-type (SPINK) gene Kazal1, exhibit a massive autophagy after amputation that rapidly compromises cell and animal survival. This excessive autophagy phenotype actually mimics that observed in the mammalian pancreas when SPINK genes are mutated, highlighting the paradigmatic value of the Hydra model system for deciphering pathological processes. Interestingly autophagy during starvation predominantly affects ectodermal epithelial cells and lead to cell survival whereas Kazal1(RNAi)-induced autophagy is restricted to endodermal digestive cells that rapidly undergo cell death. This indicates that distinct regulations that remain to be identified, are at work in these two contexts. Cnidarian express orthologs for most components of the autophagy and TOR pathways suggesting evolutionarily-conserved roles during starvation

Head regeneration in wild-type hydra requires de novo neurogenesis

Because head regeneration occurs in nerve-free hydra mutants, neurogenesis was regarded as dispensable for this process. Here, in wild-type hydra, we tested the function of the ParaHox gsx homolog gene, cnox-2, which is a specific marker for bipotent neuronal progenitors, expressed in cycling interstitial cells that give rise to apical neurons and gastric nematoblasts (i.e. sensory mechanoreceptor precursors). cnox-2 RNAi silencing leads to a dramatic downregulation of hyZic, prdl-a, gsc and cnASH, whereas hyCOUP-TF is upregulated. cnox-2 indeed acts as an upstream regulator of the neuronal and nematocyte differentiation pathways, as cnox-2(-) hydra display a drastic reduction in apical neurons and gastric nematoblasts, a disorganized apical nervous system and a decreased body size. During head regeneration, the locally restricted de novo neurogenesis that precedes head formation is cnox-2 dependent: cnox-2 expression is induced in neuronal precursors and differentiating neurons that appear in the regenerating tip; cnox-2 RNAi silencing reduces this de novo neurogenesis and delays head formation. Similarly, the disappearance of cnox-2(+) cells in sf-1 mutants also correlates with head regeneration blockade. Hence in wild-type hydra, head regeneration requires the cnox-2 neurogenic function. When neurogenesis is missing, an alternative, slower and less efficient, head developmental program is possibly activated