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  • Open access
  • 22 Reads
Regulation of extrasynaptic glutamatergic signaling by polysialylated NCAM in health and disease

The neural cell adhesion molecule NCAM is known to mediate cell-cell and cell-extracellular matrix (ECM) adhesion via homophilic and heterophilic interactions. During brain development, NCAM and associated glycan, polysialic acid (polySia), play important roles in cell migration, proliferation, neurite outgrowth and fasciculation, and synaptogenesis. In the adult rodent brain, NCAM regulates synaptic plasticity, learning, and memory. Dysregulated cortical expression of NCAM and polySia have been found in Alzheimer’s disease and schizophrenia. Our data demonstrate the essential role of polySia-NCAM in the balancing of signaling through synaptic/extrasynaptic NMDA receptors and highlight the therapeutic potential of short polySia fragments to restrain GluN1/GluN2B-mediated signaling in several animal models of neurological and psychiatric diseases.

  • Open access
  • 19 Reads
Mesoderm subset derived from human pluripotent stem cells from diabetic and nondiabetics improve retinal pathology in a model of type 2 diabetes.

Human induced pluripotent stem cells (hiPSCs) isolated from diabetics and controls were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. These cells have robust proliferative potential and ability to differentiate into vascular wall derived reparative cells, called endothelial colony-forming cell (ECFCs). These cells incorporate into blood vessels when implanted subcutaneously into the flank of non-obese diabetic/severe combined immunodeficient mice. KNA cells+ of diabetic or nondiabetic origin when injected into the vitreous of diabetic mice with retinopathy incorporated into blood vessels and increased the number of perfused capillaries in the damaged retina. Transcriptomic analysis demonstrated that differentiation of hiPSCs derived from diabetics into KNA+ cells reprogram diabetic cells to a pattern like KNA+ cells derived from nondiabetic hiPSCs. Proteomic studies performed on retinas of diabetic mice injected with either control or diabetic donor-derived KNA+ cells showed correction of aberrant signaling in diabetic retinas toward normal healthy retina. These studies support that KNA+ cells and ECFCs can correct vascular dysfunction in diabetic mice.

  • Open access
  • 38 Reads
Compartmentalization and trafficking in endoplasmic reticulum protein quality control

Following translocation into the rough endoplasmic reticulum (ER), secretory proteins undergo a series of folding, maturation, compartmentalization and trafficking events. These are finely tuned to avoid misfolded protein accumulation and the consequent ER stress. Misfolded proteins and components of the ER quality control and ER associated degradation (ERAD) machineries concentrate in mammalian cells in the pericentriolar ER-derived quality control compartment (ERQC), a staging ground for ERAD. We have recently determined that, surprisingly, trafficking to the ERQC and delivery to ERAD are dependent on COPII-coated vesicle transport and that they can be retrieved to the peripheral ER in COPI-coated vesicles.

  • Open access
  • 36 Reads
Apremilast is protective in pemphigus by a Plakoglobin-dependent stabilization of keratinocyte adhesion
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Desmosomes provide adhesive strength to tissues constantly exposed to mechanical stress such as the heart and the epidermis. The importance of desmosomes is reflected by several severe diseases, in which desmosomal adhesion is compromised. Among them, pemphigus vulgaris, a bullous autoimmune disease, in which autoantibodies directed against the desmosomal cadherins desmoglein (Dsg) 1 and 3 cause loss of intercellular adhesion clinically manifested as flaccid blisters of the skin and mucous membranes. At present, therapies focus on suppression of autoantibody formation. However, especially for the acute phase of the disease an additional treatment paradigm directly stabilizing keratinocyte adhesion would fulfill an unmet medical need. We here demonstrate that apremilast, a phosphodiesterase 4 inhibitor used in psoriasis, is protective against pemphigus autoantibody-induced loss of intercellular adhesion in human keratinocytes in vitro. In addition, apremilast abrogates pemphigus autoantibody-induced blistering in ex-vivo human epidermis and in vivo in mice. In parallel, apremilast inhibits keratin retraction as well as desmosome splitting. Further, apremilast induces phosphorylation of plakoglobin at serine 665 - a mechanisms which is known to stabilize cardiomyocyte cohesion. Thus, we established a plakoglobin phospho-deficient mouse model. Phospho-deficient mice reveal a fragile epidermis with altered organization of keratin filaments and desmosomal cadherins. In keratinocytes derived from these mice, intercellular adhesion is impaired and not rescued by apremilast. These data identify an unreported mechanism of desmosome regulation and propose that apremilast stabilizes keratinocyte adhesion via keratin anchorage in pemphigus. Thus, Apremilast may serve as treatment option during the acute phase in pemphigus

  • Open access
  • 18 Reads
Development of therapies for the two sides of the cornea

The clearness of the cornea is essential for visual acuity and is guaranteed by limbal stem cell regeneration of the outer specialized epithelium and by the integrity of the inner corneal endothelium.

Limbal Stem Cell Deficiency (LSCD) leads to multiple corneal abnormalities including visual loss. Autologous Cultivated Limbal Stem Cell Transplantation is the first stem cell treatment for LSCD, approved at EU level. Here we present the results from the first EU multicenter clinical trial on EU citizens and a following step for the future restoration of other corneal areas, specifically the corneal endothelium.

  • Open access
  • 19 Reads
Keeping mitochondria in shape: a matter of cell life and death

In the last years, mitochondrial ultrastructural and morphological changes have been implied in the control of several physiological and pathological changes, including the progression of apoptosis, inflammation, differentiation, tumorigenesis. However, the role of mitochondrial dynamics in the control of complex cellular cues and in response to reversible and irreversible cellular damage is not yet clarified. We will overview the key experiments that shed light on the role of mitochondrial shape and ultrastructure in cell physiology, pathology and in disease and offer a personal perspective on the missing pieces of the puzzle that await to be studied.

  • Open access
  • 22 Reads
The Rab11 family controls signalling to the cytoskeleton for cell migration and invasion
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Endocytic recycling controls the return of internalised cargos to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargos to regulate cell behaviour. We have previously shown that Rab11 regulates recycling of integrins to promote cancer cell migration and invasion, at least in part by controlling RhoGTPase activity at the leading edge, but the mechanisms that underpin cytoskeleton regulation by Rab11 family members are still unclear.

We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing robust protein-protein interaction networks of well characterised and new cargos and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion, and we demonstrate that several of these new Rab11 and Rab25 associated proteins are required for efficient cancer cell migration in 3D-matrix.

Rab11 and Rab25 vesicles are found at the perinuclear recycling compartment but also in the tips of protrusions in cells moving in 3D matrix. This leads us to speculate that these recycling pathways deliver cargoes to directly promote protrusion formation and extension. To test this, we established a magneto-genetic approach to physically re-localise Rab25 vesicles in cells in 2D and 3D matrix. Using this technique, we are able to show that repositioning of Rab25 vesicles to the cell cortex promotes the formation of protrusions in a manner dependent on actin polymerising proteins formins, but not Arp2/3. Together, these data reveal a direct role for Rab11 family members in directing cytoskeletal signalling to promote cancer cell invasion.

  • Open access
  • 23 Reads
Deciphering the crosstalk between cancer stem cells and the tumor immune microenvironment

Immune checkpoint blockade therapies aim to re-establish the antitumor immune response of effector lymphocytes. Although these therapies have revolutionized cancer treatment, only a minority of patients benefits from them. Two major aspects obstacle the effectiveness of immune-checkpoint inhibitor therapies, the composition of the tumor microenvironment and the quiescence of cancer cells including cancer stem cells (CSCs). Understanding the molecular mechanisms underlying these problems is key to boost antitumor immunity.

Eukaryotic cells have evolved complex systems to decipher and respond to signals that come from the environment. One of the second messengers widely used in nature by both plant and animal cells to interpret environmental stimuli is the Ca2+ ion. In response to extracellular stimuli, the intracellular concentration of Ca2+ increases. Ca2+ sensors such as calmodulin and calcineurin (CN) are conserved in evolution from plants to humans. In plants, yeasts and fungi, CN is recognized as a stress sensor that decides on cell proliferation and survival in response to environmental signals. CN, a serine (Ser)/threonine (Thr) phosphatase, serves as an important translator of information from the local calcium rise to the effectors that direct cellular responses. For instance, it is activated in dendritic cells (DCs) downstream of Pattern Recognition Receptors (PRRs) in response to microbial stimuli and is responsible for their terminal differentiation, moreover CN activation in T and B cells is fundamental for their proliferation and differentiation. The tumor-intrinsic role of CN in controlling the formation of the tumor microenvironment, quiescence of CSCs, and the responsiveness to immune checkpoint inhibitor therapies are discussed.

  • Open access
  • 19 Reads
Implication of intra-tumor heterogeneity on colorectal cancer response to MEK inhibition

Intra-tumor heterogeneity (ITH) poses a major obstacle in cancer therapy. In colorectal cancer (CRC), mutations in the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) pathway, especially in the SMAD4 gene have been correlated with decreased overall survival and are suspected to modulate chemoresistance. We have previously shown that SMAD4R361H is associated with differential drug response towards EGFR, MEK and PI3K inhibitors.

Here, we analyzed the mechanistic role of SMAD4R361H using oncoproteomics in CRISPR-engineered SMAD4R361H, CRC patient-derived organoids (PD3D®). Utilizing DigiWest® multiplex protein profiling analysis, we confirmed stronger response to MEK inhibition in organoids harboring SMAD4R361H as compared to SMAD4wt PD3D. After 24 hours of incubation with 0.03 µM trametinib, we observed a more pronounced decrease in proliferation markers, such as cyclin B1 and aurora kinase A in SMAD4R361H cells. Interestingly, there were no noticeable accumulation of caspases 3 and 9 in any organoid culture, however there was a conspicuous trend in accumulation of Bcl-xL in presence of SMAD4R361H. To understand the underlaying mechanism of such a discrepancy, we analyzed the protein levels and phosphorylation status of other SMADs, as SMAD4R361H disrupts TGF-β/BMP signal transduction. Out of all SMADs, only SMAD5 showed significant changes in protein level and phosphorylation status in response to the treatment only in SMAD4wt organoids. As previously published, BMP signaling promotes cancer cell proliferation and tumor growth. It is plausible to assume that functional loss of SMAD4 and thus loss of SMAD5 signaling renders SMAD4R361H subpopulation of cells more sensitive to MEK inhibitors. Loss of SMAD4 was previously shown to promote chemoresistance and associate with higher recurrence rate in colorectal cancer.

The heterogenic landscape of mutated SMAD4 within the same tumor, in this light, can give rise to multi-drug resistant disease.