tRNA-derived fragments in T lymphocyte-beta cell crosstalk and in type 1 diabetes pathogenesis in NOD mice.

AIMS/HYPOTHESIS: tRNAs play a central role in protein synthesis. Besides this canonical function, they were recently found to generate non-coding RNA fragments (tRFs) regulating different cellular activities. The aim of this study was to assess the involvement of tRFs in the crosstalk between immune cells and beta cells and to investigate their contribution to the development of type 1 diabetes. METHODS: Global profiling of the tRFs present in pancreatic islets of 4- and 8-week-old NOD mice and in extracellular vesicles released by activated CD4+ T lymphocytes was performed by small RNA-seq. Changes in the level of specific fragments were confirmed by quantitative PCR. The transfer of tRFs from immune cells to beta cells occurring during insulitis was assessed using an RNA-tagging approach. The functional role of tRFs increasing in beta cells during the initial phases of type 1 diabetes was determined by overexpressing them in dissociated islet cells and by determining the impact on gene expression and beta cell apoptosis. RESULTS: We found that the tRF pool was altered in the islets of NOD mice during the initial phases of type 1 diabetes. Part of these changes were triggered by prolonged exposure of beta cells to proinflammatory cytokines (IL-1ß, TNF-α and IFN-γ) while others resulted from the delivery of tRFs produced by CD4+ T lymphocytes infiltrating the islets. Indeed, we identified several tRFs that were enriched in extracellular vesicles from CD4+/CD25- T cells and were transferred to beta cells upon adoptive transfer of these immune cells in NOD.SCID mice. The tRFs delivered to beta cells during the autoimmune reaction triggered gene expression changes that affected the immune regulatory capacity of insulin-secreting cells and rendered the cells more prone to apoptosis. CONCLUSIONS/INTERPRETATION: Our data point to tRFs as novel players in the crosstalk between the immune system and insulin-secreting cells and suggest a potential involvement of this novel class of non-coding RNAs in type 1 diabetes pathogenesis. DATA AVAILABILITY: Sequences are available from the Gene Expression Omnibus (GEO) with accession numbers GSE242568 and GSE256343.

Open and remotely accessible Neuroplatform for research in wetware computing.

Wetware computing and organoid intelligence is an emerging research field at the intersection of electrophysiology and artificial intelligence. The core concept involves using living neurons to perform computations, similar to how Artificial Neural Networks (ANNs) are used today. However, unlike ANNs, where updating digital tensors (weights) can instantly modify network responses, entirely new methods must be developed for neural networks using biological neurons. Discovering these methods is challenging and requires a system capable of conducting numerous experiments, ideally accessible to researchers worldwide. For this reason, we developed a hardware and software system that allows for electrophysiological experiments on an unmatched scale. The Neuroplatform enables researchers to run experiments on neural organoids with a lifetime of even more than 100 days. To do so, we streamlined the experimental process to quickly produce new organoids, monitor action potentials 24/7, and provide electrical stimulations. We also designed a microfluidic system that allows for fully automated medium flow and change, thus reducing the disruptions by physical interventions in the incubator and ensuring stable environmental conditions. Over the past three years, the Neuroplatform was utilized with over 1,000 brain organoids, enabling the collection of more than 18 terabytes of data. A dedicated Application Programming Interface (API) has been developed to conduct remote research directly via our Python library or using interactive compute such as Jupyter Notebooks. In addition to electrophysiological operations, our API also controls pumps, digital cameras and UV lights for molecule uncaging. This allows for the execution of complex 24/7 experiments, including closed-loop strategies and processing using the latest deep learning or reinforcement learning libraries. Furthermore, the infrastructure supports entirely remote use. Currently in 2024, the system is freely available for research purposes, and numerous research groups have begun using it for their experiments. This article outlines the system's architecture and provides specific examples of experiments and results.

Small RNAs derived from tRNA fragmentation regulate the functional maturation of neonatal ß cells.

tRNA-derived fragments (tRFs) are an emerging class of small non-coding RNAs with distinct cellular functions. Here, we studied the contribution of tRFs to the regulation of postnatal ß cell maturation, a critical process that may lead to diabetes susceptibility in adulthood. We identified three tRFs abundant in neonatal rat islets originating from 5' halves (tiRNA-5s) of histidine and glutamate tRNAs. Their inhibition in these islets reduced ß cell proliferation and insulin secretion. Mitochondrial respiration was also perturbed, fitting with the mitochondrial enrichment of nuclear-encoded tiRNA-5HisGTG and tiRNA-5GluCTC. Notably, tiRNA-5 inhibition reduced Mpc1, a mitochondrial pyruvate carrier whose knock down largely phenocopied tiRNA-5 inhibition. tiRNA-5HisGTG interactome revealed binding to Musashi-1, which was essential for the mitochondrial enrichment of tiRNA-5HisGTG. Finally, tiRNA-5s were dysregulated in the islets of diabetic and diabetes-prone animals. Altogether, tiRNA-5s represent a class of regulators of ß cell maturation, and their deregulation in neonatal islets may lead to diabetes susceptibility in adulthood.

Role of microRNA in pancreatic beta cell function.

MicroRNA (miRNAs) are small non-coding RNA involved in gene expression regulation. Emerging evidences identify miRNAs as key regulators of beta cell physiology. Their role in fine-tuned gene expression regulation is crucial in the differentiation of insulin-producing cells and contributes to the acquisition and management of their unique phenotype. Dysregulation of miRNA expression causes beta cell dysfunction and promotes the development of different forms of diabetes mellitus.

Circular RNAs as Novel Regulators of ß-Cell Functions under Physiological and Pathological Conditions.

Circular RNAs (circRNAs) constitute a large class of non-coding RNAs characterized by a covalently closed circular structure. They originate during mRNA maturation through a modification of the splicing process and, according to the included sequences, are classified as Exonic, Intronic, or Exonic-Intronic. CircRNAs can act by sequestering microRNAs, by regulating the activity of specific proteins, and/or by being translated in functional peptides. There is emerging evidence indicating that dysregulation of circRNA expression is associated with pathological conditions, including cancer, neurological disorders, cardiovascular diseases, and diabetes. The aim of this review is to provide a comprehensive and updated view of the most abundant circRNAs expressed in pancreatic islet cells, some of which originating from key genes controlling the differentiation and the activity of insulin-secreting cells or from diabetes susceptibility genes. We will particularly focus on the role of a group of circRNAs that contribute to the regulation of ß-cell functions and that display altered expression in the islets of rodent diabetes models and of type 2 diabetic patients. We will also provide an outlook of the unanswered questions regarding circRNA biology and discuss the potential role of circRNAs as biomarkers for ß-cell demise and diabetes development.

A circular RNA generated from an intron of the insulin gene controls insulin secretion.

Fine-tuning of insulin release from pancreatic ß-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of ß-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa (TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding ß-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder.

Roles of Noncoding RNAs in Islet Biology.

The discovery that most mammalian genome sequences are transcribed to ribonucleic acids (RNA) has revolutionized our understanding of the mechanisms governing key cellular processes and of the causes of human diseases, including diabetes mellitus. Pancreatic islet cells were found to contain thousands of noncoding RNAs (ncRNAs), including micro-RNAs (miRNAs), PIWI-associated RNAs, small nucleolar RNAs, tRNA-derived fragments, long non-coding RNAs, and circular RNAs. While the involvement of miRNAs in islet function and in the etiology of diabetes is now well documented, there is emerging evidence indicating that other classes of ncRNAs are also participating in different aspects of islet physiology. The aim of this article will be to provide a comprehensive and updated view of the studies carried out in human samples and rodent models over the past 15 years on the role of ncRNAs in the control of α- and ß-cell development and function and to highlight the recent discoveries in the field. We not only describe the role of ncRNAs in the control of insulin and glucagon secretion but also address the contribution of these regulatory molecules in the proliferation and survival of islet cells under physiological and pathological conditions. It is now well established that most cells release part of their ncRNAs inside small extracellular vesicles, allowing the delivery of genetic material to neighboring or distantly located target cells. The role of these secreted RNAs in cell-to-cell communication between ß-cells and other metabolic tissues as well as their potential use as diabetes biomarkers will be discussed. © 2020 American Physiological Society. Compr Physiol 10:893-932, 2020.

The miRNAs miR-211-5p and miR-204-5p modulate ER stress in human beta cells.

miRNAs are a class of small non-coding RNAs that regulate gene expression. Type 1 diabetes is an autoimmune disease characterized by insulitis (islets inflammation) and pancreatic beta cell destruction. The pro-inflammatory cytokines interleukin 1 beta (IL1B) and interferon gamma (IFNG) are released during insulitis and trigger endoplasmic reticulum (ER) stress and expression of pro-apoptotic members of the BCL2 protein family in beta cells, thus contributing to their death. The nature of miRNAs that regulate ER stress and beta cell apoptosis remains to be elucidated. We have performed a global miRNA expression profile on cytokine-treated human islets and observed a marked downregulation of miR-211-5p. By real-time PCR and Western blot analysis, we confirmed cytokine-induced changes in the expression of miR-211-5p and the closely related miR-204-5p and downstream ER stress related genes in human beta cells. Blocking of endogenous miRNA-211-5p and miR-204-5p by the same inhibitor (it is not possible to block separately these two miRs) increased human beta cell apoptosis, as measured by Hoechst/propidium Iodide staining and by determination of cleaved caspase-3 activation. Interestingly, miRs-211-5p and 204-5p regulate the expression of several ER stress markers downstream of PERK, particularly the pro-apoptotic protein DDIT3 (also known as CHOP). Blocking CHOP expression by a specific siRNA partially prevented the increased apoptosis observed following miR-211-5p/miR-204-5p inhibition. These observations identify a novel crosstalk between miRNAs, ER stress and beta cell apoptosis in early type 1 diabetes.

Prolactin protects against cytokine-induced beta-cell death by NFκB and JNK inhibition.

Type 1 diabetes is caused by an autoimmune assault that induces progressive beta-cell dysfunction and dead. Pro-inflammatory cytokines, such as interleukin 1 beta (IL1B), tumor necrosis factor (TNF) and interferon gamma (IFNG) contribute for beta-cell death, which involves the activation of the nuclear factor kappa B (NFκB) and c- Jun N-terminal kinase (JNK). Prolactin (PRL), a physiological mediator for beta-cell proliferation, was shown to protect beta cells against cytokines pro-apoptotic effects. We presently investigated the mechanisms involved in the protective effects of prolactin against cytokine-induced beta-cell death. The findings obtained indicate that STAT3 activation is involved in the anti-apoptotic role of PRL in rat beta cells. PRL prevents the activation of JNK via AKT and promotes a shift from expression of pro- to anti-apoptotic proteins downstream of the JNK cascade. Furthermore, PRL partially prevents the activation of NFκB and the transcription of its target genes IkBa, Fas, Mcp1, A20 and Cxcl10 and also decreases NO production. On the other hand, the pro-survival effects of PRL do not involve modulation of cytokine-induced endoplasmic reticulum stress. These results suggest that the beneficial effects of PRL in beta cells involve augmentation of anti-apoptotic mechanisms and, at the same time, reduction of pro-apoptotic effectors, rendering beta cells better prepared to deal with inflammatory insults. The better understanding of the pro-survival mechanisms modulated by PRL in beta cells can provide tools to prevent cell demise during an autoimmune attack or following islet transplantation.

Ubiquitin D Regulates IRE1α/c-Jun N-terminal Kinase (JNK) Protein-dependent Apoptosis in Pancreatic Beta Cells.

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1ß and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells.

ER stress and the decline and fall of pancreatic beta cells in type 1 diabetes.

Components of the unfolded protein response (UPR) modulate beta cell inflammation and death in early type 1 diabetes (T1D). The UPR is a mechanism by which cells react to the accumulation of misfolded proteins in the endoplasmic reticulum (ER). It aims to restore cellular homeostasis, but in case of chronic or overwhelming ER stress the persistent activation of the UPR triggers apoptosis, contributing to the loss of beta cells in both T1D and type 2 diabetes. It remains to be determined how and why the transition from 'physiological' to 'pathological' UPR takes place. A key component of the UPR is the ER transmembrane protein IRE1α (inositol-requiring enzyme 1α). IRE1α activity is modulated by both intra-ER signals and by the formation of protein complexes at its cytosolic domain. The amplitude and duration of IRE1α signaling is critical for the transition between the adaptive and cell death programs, with particular relevance for the activation of the pro-apoptotic c-Jun N-terminal kinase (JNK) in beta cells. In the present review we discuss the available information on IRE1α-regulating proteins in beta cells and their downstream targets, and the important differences observed between cytokine-induced UPR in human and rodent beta cells.

Cytokines induce endoplasmic reticulum stress in human, rat and mouse beta cells via different mechanisms.

AIMS/HYPOTHESIS: Proinflammatory cytokines contribute to beta cell damage in type 1 diabetes in part through activation of endoplasmic reticulum (ER) stress. In rat beta cells, cytokine-induced ER stress involves NO production and consequent inhibition of the ER Ca(2+) transporting ATPase sarco/endoplasmic reticulum Ca(2+) pump 2 (SERCA2B). However, the mechanisms by which cytokines induce ER stress and apoptosis in mouse and human pancreatic beta cells remain unclear. The purpose of this study is to elucidate the role of ER stress on cytokine-induced beta cell apoptosis in these three species and thus solve ongoing controversies in the field. METHODS: Rat and mouse insulin-producing cells, human pancreatic islets and human EndoC-ßH1 cells were exposed to the cytokines IL-1ß, TNF-α and IFN-γ, with or without NO inhibition. A global comparison of cytokine-modulated gene expression in human, mouse and rat beta cells was also performed. The chemical chaperone tauroursodeoxycholic acid (TUDCA) and suppression of C/EBP homologous protein (CHOP) were used to assess the role of ER stress in cytokine-induced apoptosis of human beta cells. RESULTS: NO plays a key role in cytokine-induced ER stress in rat islets, but not in mouse or human islets. Bioinformatics analysis indicated greater similarity between human and mouse than between human and rat global gene expression after cytokine exposure. The chemical chaperone TUDCA and suppression of CHOP or c-Jun N-terminal kinase (JNK) protected human beta cells against cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: These observations clarify previous results that were discrepant owing to the use of islets from different species, and confirm that cytokine-induced ER stress contributes to human beta cell death, at least in part via JNK activation.

Clic4, a novel protein that sensitizes ß-cells to apoptosis.

OBJECTIVES: Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic ß-cell apoptosis. METHODS: We used ßTC-tet cells and islets from ß-cell specific Clic4 knockout mice (ßClic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis. RESULTS: We show that cytokines increased Clic4 expression in ßTC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in ßTC-tet cells or its genetic inactivation in islets ß-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in ßTC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets ß-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from ßTC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation. CONCLUSIONS: Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes ß-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad.

A combined "omics" approach identifies N-Myc interactor as a novel cytokine-induced regulator of IRE1 protein and c-Jun N-terminal kinase in pancreatic beta cells.

Type 1 diabetes is an autoimmune disease with a strong inflammatory component. The cytokines interleukin-1ß and interferon-γ contribute to beta cell apoptosis in type 1 diabetes. These cytokines induce endoplasmic reticulum stress and the unfolded protein response (UPR), contributing to the loss of beta cells. IRE1α, one of the UPR mediators, triggers insulin degradation and inflammation in beta cells and is critical for the transition from "physiological" to "pathological" UPR. The mechanisms regulating inositol-requiring protein 1α (IRE1α) activation and its signaling for beta cell "adaptation," "stress response," or "apoptosis" remain to be clarified. To address these questions, we combined mammalian protein-protein interaction trap-based IRE1α interactome and functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines to identify novel cytokine-induced regulators of IRE1α. Based on this approach, we identified N-Myc interactor (NMI) as an IRE1α-interacting/modulator protein in rodent and human pancreatic beta cells. An increased expression of NMI was detected in islets from nonobese diabetic mice with insulitis and in rodent or human beta cells exposed in vitro to the pro-inflammatory cytokines interleukin-1ß and interferon-γ. Detailed mechanistic studies demonstrated that NMI negatively modulates IRE1α-dependent activation of JNK and apoptosis in rodent and human pancreatic beta cells. In conclusion, by using a combined omics approach, we identified NMI induction as a novel negative feedback mechanism that decreases IRE1α-dependent activation of JNK and apoptosis in cytokine-exposed beta cells

MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway.

Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)-anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited by small interfering RNA knockdown of MyRIP, which also reduced cAMP-mediated hormone secretion. Demonstrating the importance of this phosphorylation, nonphosphorylatable and phosphomimic Rph-3A mutants significantly altered hormone release when PKA was activated. These data suggest that MyRIP only forms a functional protein complex with BR-MyoVa on SGs when cAMP is elevated and under this condition facilitates phosphorylation of SG-associated proteins, which in turn can enhance secretion.

Molecular mechanism of myosin Va recruitment to dense core secretory granules.

The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

The many faces of S100B protein: when an extracellular factor inactivates its own receptor and activates another one.

The Ca(2+)-binding protein of the EF-hand type, S100B, is an intracellular regulator and an extracellular signal. Within cells S100B interacts with several proteins thereby regulating energy metabolism, Ca2+ homeostasis, protein phosphorylation and degradation, and cell locomotion, proliferation and differentiation. Once secreted/released, S100B exerts autocrine and paracrine effects on responsive cells by engaging the receptor for advanced glycation end products. However, recent evidence suggests that S100B might also activate basic fibroblast growth factor receptor 1 via prior binding to basic fibroblast growth factor.

S100B Protein Regulates Astrocyte Shape and Migration via Interaction with Src Kinase: IMPLICATIONS FOR ASTROCYTE DEVELOPMENT, ACTIVATION, AND TUMOR GROWTH.

S100B is a Ca(2+)-binding protein of the EF-hand type that is abundantly expressed in astrocytes and has been implicated in the regulation of several intracellular activities, including proliferation and differentiation. We show here that reducing S100B levels in the astrocytoma cell line GL15 and the Müller cell line MIO-M1 by small interference RNA technique results in a rapid disassembly of stress fibers, collapse of F-actin onto the plasma membrane and reduced migration, and acquisition of a stellate shape. Also, S100B-silenced GL15 and MIO-M1 Müller cells show a higher abundance of glial fibrillary acidic protein filaments, which mark differentiated astrocytes, compared with control cells. These effects are dependent on reduced activation of the phosphatidylinositol 3-kinase (PI3K) downstream effectors, Akt and RhoA, and consequently elevated activity of GSK3beta and Rac1 and decreased activity of the RhoA-associated kinase. Also, rat primary astrocytes transiently down-regulate S100B expression when exposed to the differentiating agent dibutyryl cyclic AMP and re-express S100B at later stages of dibutyryl cyclic AMP-induced differentiation. Moreover, reducing S100B levels results in a remarkably slow resumption of S100B expression, suggesting the S100B might regulate its own expression. Finally, we show that S100B interacts with Src kinase, thereby stimulating the PI3K/Akt and PI3K/RhoA pathways. These results suggest that S100B might contribute to reduce the differentiation potential of cells of the astrocytic lineage and participate in the astrocyte activation process in the case of brain insult and in invasive properties of glioma cells.

S100B's double life: intracellular regulator and extracellular signal.

The Ca2+-binding protein of the EF-hand type, S100B, exerts both intracellular and extracellular functions. Recent studies have provided more detailed information concerning the mechanism(s) of action of S100B as an intracellular regulator and an extracellular signal. Indeed, intracellular S100B acts as a stimulator of cell proliferation and migration and an inhibitor of apoptosis and differentiation, which might have important implications during brain, cartilage and skeletal muscle development and repair, activation of astrocytes in the course of brain damage and neurodegenerative processes, and of cardiomyocyte remodeling after infarction, as well as in melanomagenesis and gliomagenesis. As an extracellular factor, S100B engages RAGE (receptor for advanced glycation end products) in a variety of cell types with different outcomes (i.e. beneficial or detrimental, pro-proliferative or pro-differentiative) depending on the concentration attained by the protein, the cell type and the microenvironment. Yet, RAGE might not be the sole S100B receptor, and S100B's ability to engage RAGE might be regulated by its interaction with other extracellular factors. Future studies using S100B transgenic and S100B null mice might shed more light on the functional role(s) of the protein.

GL15 and U251 glioblastoma-derived human cell lines are peculiarly susceptible to induction of mitotic death by very low concentrations of okadaic acid.

The challenging possibility of selectively inducing mitotic death in tumor cells by combining genotoxic agents with the inhibition of G2 checkpoints of the cell cycle is the subject of intensive investigation. We show that very low concentrations (3.5 and 5 nM) of okadaic acid induce mitotic death in two glioblastoma cell lines, in the absence of genotoxic agents. At the concentrations used, the main target of okadaic acid action is protein phosphatase 2A (PP2A), an enzyme deeply involved in the negative control of cell-cycle progression. The peculiar susceptibility of glioblastoma cells to induction of mitotic death by very low concentrations of okadaic acid must be related to an impairment of PP2A activity and to a specific deficiency in some cell-cycle checkpoints. In addition to its ability to induce abnormal mitoses in actively proliferating glioblastoma cells, okadaic acid possesses the ability to force semi-confluent glioblastoma cells to the M phase of the cell cycle, where they show the same abnormalities observed in actively proliferating glioblastoma cells. In semi-confluent cells the induction of mitotic death involves the activity of both the extracellular signal regulated kinases (ERKs) and the M-phase promoting factor: okadaic acid overstimulates ERK activity, and PD98059 (inhibitor of ERK activation) as well as roscovitine (S)-isomer (specific inhibitor of M-phase promoting factor activity) counteract the induction of mitotic death. Our results show that, without the use of genotoxic agents, it is possible to induce mitotic death in glioblastoma cells by activating the same uncontrolled pathways responsible for the uncontrolled proliferation.