Biology subjects

Bultynck, G.

Publications and source records attributed to Bultynck, G..

8 recordsLinked to original sources

bioRxiv2026

TRPA1 channel activation by synthetic lipid nanoparticles

TRPA1 is a polymodal ion channel receptor known for its role in nociception. TRPA1 can be activated by local mechanical perturbations in the surrounding plasma membrane (PM) by molecules that insert in the lipid bilayer. Here, we tested whether TRPA1 function can be modulated by lipid nanoparticles (LNPs) while interacting with the target cell plasma membrane. We found that LNP induce irregular Ca2+ transients in heterologous and native TRPA1-expressing cells, which may reflect stochastic LNP-PM interactions. By using different cell types and applying selective and non-selective TRPA1 inhibitors, we revealed that the cytosolic [Ca2+] is elevated transients arise as a result through multiple mechanisms: TRPA1-dependent Ca2+ influx, TRPA1-independent Ca2+ influx, and Ca2+ mobilization from the endoplasmic reticulum. Our results describe a novel, non-canonical TRPA1 activation mechanism by LNPs, that may be relevant in the context of the development of cancer and nasal vaccines.

pharmacology and toxicology↗

bioRxiv2024

Oncogenic GNAQ/11-induced remodeling of the IP3/Calcium signaling pathway protects Uveal Melanoma against Calcium-driven cell death

Despite being considered a rare tumor, uveal melanoma (UVM) is the most common adult intraocular malignancy. With a poor prognosis and limited treatment options, up to 50% of patients develop metastases, primarily in the liver. A range of mutations and chromosomal aberrations with significant prognostic value has been associated with UVM pathogenesis. The most frequently mutated genes are GNAQ and GNA11, which encode the subunits of Gq proteins and are described as driver mutations that activate multiple signaling cascades involved in cell growth and proliferation. Directly downstream of Gq/11 activation, PLC{beta} engagement leads to sustained production of DAG and IP3. While the DAG/PKC/RasGRP3/MAPK signaling branch has been identified as an essential component of UVM unregulated proliferation, the role of IP3-mediated signals has been largely overlooked. Here, we demonstrate that, whilst maintaining Ca{superscript 2} homeostasis, UVM cells have developed a decoupling mechanism between IP3 and ER Ca{superscript 2} release by altering IP3 receptor (IP3R) expression. This correlation was observed in human UVM tumors, where IP3Rs were found to be downregulated. Critically, when IP3R3 expression was restored, UVM cells exhibited an increased tendency to undergo spontaneous cell death and became more sensitive to pro-apoptotic modulators of IP3R-mediated Ca{superscript 2} signaling, such as staurosporine and the Bcl2-IP3R disrupter peptide BIRD2. Finally, inhibition of the Gq/11 signaling pathway revealed that IP3R expression is negatively regulated by GNAQ/11 oncogenic activation. Hence, we demonstrated that by remodeling IP3R expression, GNAQ/11 oncogenes protect UVM cells against IP3-triggered Ca{superscript 2} overload and cell death. Therefore, the GNAQ/11 pathway not only drives proliferation through DAG activity but also provides a protective mechanism to evade IP3/Ca{superscript 2}-mediated cell death. These dual functions could potentially be exploited in novel combinatorial therapeutic strategies to effectively block UVM cell proliferation while simultaneously sensitizing them to cell death.

cancer biology↗

bioRxiv2024

IP3 receptor depletion in a spontaneous canine model of Charcot-Marie-Tooth disease 1J with amelogenesis imperfecta

Inositol 1,4,5-trisphosphate receptors (IP3R) mediate Ca2+ release from intracellular stores, contributing to complex regulation of numerous physiological responses. The involvement of the three IP3R genes (ITPR1, ITPR2 and ITPR3) in inherited human diseases has started to shed light on the essential roles of each receptor in different human tissues and cell types. Variants in the ITPR3 gene, which encodes IP3R3, have recently been found to cause demyelinating sensorimotor Charcot-Marie-Tooth neuropathy type 1J (CMT1J). In addition to peripheral neuropathy, immunodeficiency and tooth abnormalities are occasionally present. Here, we report the identification of a homozygous nonsense variant in the ITPR3 gene in Lancashire Heeler dogs, presenting with a severe developmental enamel defect and reduced nerve conduction velocity. We studied the primary skin fibroblasts of the affected dogs and observed that the nonsense variant in ITPR3 led to a complete absence of full-length IP3R3 protein. Unexpectedly, the protein levels of IP3R1 and IP3R2 were also markedly decreased, suggesting co-regulation. Functional Ca2+ measurements revealed reduced IP3R-mediated Ca2+ flux upon stimulation of G-protein-coupled-receptors in the affected dog fibroblasts. We were able to rescue the IP3R1 and IP3R2 depletion by proteasome inhibition but not the IP3R3 loss, which was facilitated by nonsense-mediated mRNA decay. These findings highlight the first spontaneous mammalian phenotype caused by a nonsense variant in ITPR3, leading to the loss of IP3R3. The human and canine IP3R3 proteins are highly similar, and our study suggests that the tissue involvement resulting from the receptors dysfunction is also conserved. In summary, IP3R3 is critical for enamel formation and peripheral nerve maintenance. Author summaryWe investigated pet dogs, Lancashire Heelers, with impairments in tooth development and in the nerves that regulate limb muscles. Through genetic studies of the dog pedigree, we found that the phenotypes were caused by a recessively inherited mutation in the ITPR3 gene, which encodes one of three IP3 receptors (IP3R) isoforms (IP3R3 isoform) that are needed for intracellular Ca2+ signaling. Mutated IP3R3 has been recently linked to a human inherited neuropathy called Charcot-Marie-Tooth disease type 1J, which impairs peripheral nerve function and is accompanied by immunodeficiency and abnormal teeth in some individuals. We showed that in the skin cells of the affected dogs, the full-length IP3R3 protein was completely absent, and also the protein levels of the other two IP3R isoforms (IP3R1 and IP3R2) were severely lowered. This led to impaired agonist-induced Ca2+ release and signaling. Our results demonstrate the high conservation between human and canine IP3 receptors and their significance for different tissue systems. The genetic studies now highlight that IP3R3 is vital for peripheral nerve function and enamel development.

genetics↗

bioRxiv2024

A deep phenotyping study in mouse and iPSC models to understand the role of oligodendroglia in optic neuropathy in Wolfram syndrome

Wolfram syndrome (WS) is a rare childhood disease characterized by diabetes mellitus, diabetes insipidus, blindness, deafness, neurodegeneration and eventually early death, due to autosomal recessive mutations in the WFS1 (and WFS2) gene. While it is categorized as a neurodegenerative disease, it is increasingly becoming clear that other cell types besides neurons may be affected and contribute to the pathogenesis. MRI studies in patients and phenotyping studies in WS rodent models indicate white matter/myelin loss, implicating a role for oligodendroglia in WS-associated neurodegeneration. In this study, we sought to determine if oligodendroglia are affected in WS and whether their dysfunction may be the primary cause of the observed optic neuropathy and brain neurodegeneration. We demonstrate that 7.5-month-old Wfs1{Delta}exon8 mice display signs of abnormal myelination and a reduced number of oligodendrocyte precursor cells (OPCs) as well as abnormal axonal conduction in the optic nerve. An MRI study of the brain furthermore revealed grey and white matter loss in the cerebellum, brainstem, and superior colliculus, as is seen in WS patients. To further dissect the role of oligodendroglia in WS, we performed a transcriptomics study of WS patient iPSC-derived OPCs and pre-myelinating oligodendrocytes. Transcriptional changes compared to isogenic control cells were found for genes with a role in ER function. However, a deep phenotyping study of these WS patient iPSC-derived oligodendroglia unveiled normal differentiation, mitochondria-associated endoplasmic reticulum (ER) membrane interactions and mitochondrial function, and no overt signs of ER stress. Overall, the current study indicates that oligodendroglia functions are largely preserved in the WS mouse and patient iPSC-derived models used in this study. These findings do not support a major defect in oligodendroglia function as the primary cause of WS, and warrant further investigation of neurons and neuron-oligodendroglia interactions as a target for future neuroprotective or -restorative treatments for WS.

neuroscience↗

bioRxiv2022

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing Mcl-1-addicted cancer cells

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). We previously demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a Bcl-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). These findings implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic Bcl-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we observed that BAPTAi alone induced apoptosis in lymphoma cell models that were highly sensitive to S63845, an Mcl-1 antagonist. BAPTAi provoked a rapid decline in Mcl-1 protein levels by inhibiting mTORC1-driven MCL-1 translation. Overexpression of nondegradable Mcl-1 rescued BAPTAi-induced cell death. We further examined how BAPTAi diminished mTORC1 activity and found that BAPTAi impaired glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, an up to now unkown effect of BAPTAi. All aforementioned effects of BAPTAi were also elicited by a BAPTAi analog with low affinity for Ca2+. Thus, our work reveals PFKFB3 inhibition as an unappreciated Ca2+-independent mechanism by which BAPTAi impairs cellular metabolism and ultimately the survival of Mcl-1-dependent cancer cells. Our work has two important implications. First, direct inhibition of PFKFB3 emerged as a key regulator of mTORC1 activity and a promising target in the treatment of Mcl-1-dependent cancers. Second, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

cell biology↗

bioRxiv2022

Long-Term Culture of Patient-Derived Cardiac Organoids Recapitulated Duchenne Muscular Dystrophy Cardiomyopathy and Disease Progression

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to-date incurable. The major cause of death is dilated cardiomyopathy, however the pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived pluripotent stem cells (DMD-CO) and isogenic-corrected controls (DMD-Iso-CO) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-CO lacks initial proliferative capacity, displayed a progressive loss of -sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, the cardiomyocyte deteriorated over time, and fibrosis and adipogenesis were observed in DMD-CO. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-CO which were associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies.

cell biology↗

bioRxiv2021

The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release

Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localization and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localization is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we showed that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs demonstrated a reduced expression of the Unfolded Protein Response (UPR) markers upon ER stress and displayed reduced inositol trisphosphate receptor (IP3R)-mediated ER calcium release. Collectively, our data show that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the ER, apart from its cliche regulation of apoptosis.

cell biology↗

bioRxiv2019

Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

BackgroundCT1 is a 25 amino acid therapeutic peptide incorporating the Zonula Occludens-1 (ZO-1)-binding domain of connexin43 (Cx43) that is currently in Phase III clinical testing for healing chronic skin wounds. In preclinical studies in mice, we reported that CT1 reduces arrhythmias and improves ventricular function following cardiac injury, effects that were accompanied by increases in PKC{varepsilon} phosphorylation of Cx43 at serine 368 (pS368). In this study, we undertake a systematic characterization of the molecular mode-of-action of CT1 in mitigating the effects of ischemia reperfusion injury on ventricular contractile function.\n\nMethods and ResultsTo determine the basis of CT1-mediated increases in pS368 we undertook tandem mass spectrometry of reactants in an in vitro assay of PKC{varepsilon} phosphorylation, identifying an interaction between negatively charged amino acids in the CT1 Asp-Asp-Leu-Glu-Iso sequence and positively charged lysines (Lys345, Lys346) in a short -helical sequence (H2) within the Cx43 CT domain. In silico modeling provided further support of the specificity of this interaction, leading us to conclude that CT1 has potential to directly interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift and phosphorylation assays, we characterized a series of CT1 variant peptides, identifying sequences competent to interact with either ZO-1 PDZ2 or the Cx43 CT, but with limited or no ability to bind both polypeptides. Based on this analysis, it was found that only those peptides competent to interact with Cx43, but not ZO-1 alone, resulted in increased pS368 phosphorylation in vitro and in vivo. Moreover, in a mouse model of global ischemia reperfusion injury we determined that pre-ischemic infusion only with those peptides competent to bind Cx43 preserved left ventricular (LV) contractile function following injury. Interestingly, a short 9 amino acid (MW=1110) Cx43-binding variant of the original 25 amino acid CT1 sequence demonstrated potent LV-protecting effects when infused either before or after ischemic injury.\n\nConclusionsInteraction of CT1 with the Cx43 CT, but not ZO-1 PDZ2, explains cardioprotection mediated by this therapeutic peptide. Pharmacophores targeting the Cx43 carboxyl terminus could provide a novel translational approach to preservation of ventricular function following ischemic injury.

cell biology↗