Biology subjects

Sebastiani, G.

Publications and source records attributed to Sebastiani, G..

8 recordsLinked to original sources

bioRxiv2026

Unconventional Small Molecule MNP-021 Protects Neuronal and Glial Function from Diabetes-Associated Glucotoxicity and Neuroinflammation

BackgroundDiabetes-associated neurodegeneration is amplified by methylglyoxal (MGO)-driven dicarbonyl stress linking hyperglycemia to neuronal insulin resistance and maladaptive neuroinflammation. We tested the neuroprotective activity of MNP-021, a non-electrophilic TRPA1 modulator, in neurons and glial cells in vitro. MethodsSH-SY5Y neurons were pretreated with MNP-021 and challenged with MGO, then profiled by high-content imaging, RNA-seq, Seahorse OCR/ECAR, glycolytic stress assays and AKT/ERK/CREB immunoblotting {+/-} insulin. In parallel, HMC3 glial cells were treated with MNP-021, exposed to LPS/TNF- or A{beta}(25-35) and tested for viability and inflammatory markers by ELISA and qRT-PCR. ResultsMGO increased nucleus-to-cytoplasm area ratio by 49% and dysregulated glucose handling, increasing 2-NBDG uptake by [~]25%, with GLUT1/GLUT4 membrane redistribution; MNP-021 normalized morphology, uptake, and transporter localization without cytotoxicity up to 10 {micro}M. RNA-seq identified 754 MGO-deregulated genes, including ISR/metabolic nodes (GCK, SESN2, PHGDH/PSAT1, PCK2); MNP-021 buffered stress-induced transcription with limited baseline effects, remodeled mitochondrial redox readouts consistent with controlled ROS signaling, while improving mitochondrial content/architecture and blunting stress-evoked compensatory glycolysis. MNP-021 restored pro-survival signaling (pAKT/pERK and nuclear pCREB), including insulin responsiveness during MGO exposure. MNP-021 reduced IL-6/TNF- release while increasing IL-10 and ARG1 ([~]1.9-fold vs LPS/TNF-) in HMC3 glial cells, shifting them toward a pro-resolving IL-10/ARG1 program with reduced A{beta}(25-35)-evoked cytokine release with GLP-1 remaining very low ([≤]10 pg/mL) and not significantly increased in this system. ConclusionsMNP-021 coordinates transcriptomic restraint, transporter-level glucose handling, mitochondrial resilience, and pro-survival/pro-resolving signaling across neuron-microglia compartments, supporting TRPA1-tuned small-molecule modulation as a candidate strategy against dicarbonyl-linked neuro-metabolic stress.

physiology↗

bioRxiv2025

Small non-coding RNA profiling reveals inflammatory and mitochondrial related changes in aging β-cells and islet macrophages

Aging is accompanied by functional decline and increased senescence of pancreatic {beta}-cells. These changes may be influenced by islet-resident macrophages (iMACs) that remodel tissue in response to environmental cues. To explore the molecular mechanisms underlying {beta}-cell aging and senescence, we profiled small non-coding RNAs (sncRNAs) in FACS-sorted {beta}-cells and iMACs from 3-, 12-, and 22-month-old mouse islets or from senescence-associated {beta}gal (SA-{beta}gal) positive {beta}-cells of 8-month-old mice. Overall, senescent {beta}-cells displayed distinct sncRNA signatures that only partially overlapped with those of aging. However, several miRNAs previously found to be deregulated in obese or diabetic conditions were modulated in both aging and senescent {beta}-cells, including upregulation of miRNAs linked to inflammation. In vitro exposure to pro-inflammatory cytokines partially reproduced these profiles. Aging also reshaped the {beta}-cell tRNA-derived fragment (tRF) pool, enhancing global mitochondrial tRF levels. Interestingly, some changes in miRNAs and tRFs were {beta}-cell specific, whereas others occurred also in other aged metabolic tissues. iMACs also showed age-related sncRNA remodeling, including upregulation of anti-inflammatory miRNAs and mitochondrial tRFs, suggesting adaptive immune reprogramming. Together, these data reveal a profound, coordinated reshaping of the sncRNA landscape in {beta}-cells and iMACs during aging, offering new insights into molecular mechanisms driving age-related islet dysfunction.

molecular biology↗

bioRxiv2025

5'tRNA-derived fragments modulate β-cell homeostasis and islet macrophage activation in type 2 diabetes

During obesity and type 2 diabetes, pancreatic {beta}-cells face chronic environmental stress, while islet-resident macrophages (iMACs) undergo metabolic reprogramming that exacerbates {beta}-cell dysfunction. Stress-induced cleavage of transfer RNAs (tRNAs) generates tRNA-derived fragments (tRFs), whose role in this context is not fully understood. We identify elevated levels of 5tRFGlu(CTC) and 5tRFGly(GCC) in {beta}-cells and iMACs from db/db mice and in islets from type 2 diabetic patients. Notably, 5tRFGlu(CTC) is also induced under prediabetic conditions and inversely correlates with insulin secretion. Lipotoxic stress triggers their production via Angiogenin-mediated cleavage. Blocking 5tRFGlu(CTC) in islets protects against {beta}-cell apoptosis and restores insulin secretion under palmitate stress. Using a {beta}-cell/macrophage co-culture system, we show that {beta}-cell contact shapes a unique macrophage phenotype (iMAC-like) that shifts upon palmitate exposure--recapitulating in vivo observations. Inhibiting 5tRFGlu(CTC) in iMAC-like cells prevents this activation switch, reduces {beta}-cell stress, and improves insulin secretion. Mechanistically, 5tRFGlu(CTC) interacts with RNA-binding proteins to regulate transcriptional and post-transcriptional pathways linked to immune activation, extracellular matrex remodeling, neurogenesis, and oxidative stress. Our study identifies 5tRFs as key mediators of islet microenvironment remodeling in diabetes, offering new insights into intercellular stress signaling in metabolic disease.

cell biology↗

bioRxiv2023

Comprehensive IsomiR sequencing profile of human pancreatic islets and EndoC-βH1 beta-cells

Aims/HypothesisMiRNAs play a crucial role in regulating the islet transcriptome, influencing beta cell functions and pathways. Emerging evidence suggests that during biogenesis a single miRNA locus can generate various sequences, known as isomiRNAs (isomiRs). However, a comprehensive profiling analysis of isomiRs in human pancreatic islets and beta cells is still lacking. This study aims to unveil the isomiRs expression profile in Laser Capture Microdissected (LCM) human pancreatic islets (HI) from non-diabetic donors and in the human beta cell line EndoC-{beta}H1, in order to shed light on novel molecular mechanisms governing beta cell function. MethodsRNA was extracted from LCM HI deriving from n=19 non-diabetic donors and from EndoC-{beta}H1 beta cells. Small RNA-seq was performed. Data were processed with the sRNAbench online pipeline for miRNAs/isomiRs quantification. Results were further validated using an external miRNA-seq database (isomiRdb). ResultsIn both HI and EndoC-{beta}H1, isomiRs accounted for a substantial proportion of total miRNA reads (HI: 59.4+/-1.9%; EndoC-{beta}H1: 43.8+/-0.6%). Among isomiRs, the most prevalent types were 3-end modifications, including trimming (HI=71.8+/-2.8%; EndoC-{beta}H1=55.8+/-1.0%) and extension (HI: 12.1+/-1.9%; EndoC-{beta}H1: 17.4+/-0.9%), followed by non-templated addition (HI: 9.8+/-0.9%; EndoC-{beta}H1: 14.0+/-1.2%). The analysis of the composition of the n=10 most expressed miRNAs highlighted a significant contribution of reads assigned to isomiRs. For instance, the most abundant miRNA, miR-375-3p, resulted from 59.7+/-2.4% of canonical and 40.3+/-2.4% of isomiRs in EndoC-{beta}H1 and from 45.3+/-2.0% of canonical and 54.7+/-2.0% of isomiR reads in HI. Interestingly, miR-7-5p, a beta cell-specific miRNA, was predominantly expressed as an isomiR both in EndoC-{beta}H1 (65.3+/-2.7%) and in HI (82.4+/-1.4%). To identify a reliable beta cell isomiR signature, common sequences detected in HI and EndoC-{beta}H1 were filtered based on their contribution to total miRNA expression, ultimately resulting in a set of 46 isomiRs. The expression of the isomiR signature in beta cells was further evaluated using an external database, isomiRdb, which contains small-RNA sequencing data from 99 different human cell types. This analysis revealed a significant enrichment of 11 out of the 46 isomiRs in beta cells compared to other cell types. The signature was functionally characterized through regression analysis with clinical and metabolic parameters related to beta cell function in non-diabetic individuals, demonstrating a significant negative correlation between basal insulin secretion and isomiR-411-5p, but not with its corresponding canonical miRNA. Conclusion/InterpretationThis study provides a comprehensive profile of isomiR expression in pancreatic islets and beta cells, highlighting the potential significance of isomiRs as novel regulators of beta cell function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/566223v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@145f36borg.highwire.dtl.DTLVardef@1701433org.highwire.dtl.DTLVardef@13e5866org.highwire.dtl.DTLVardef@1dbe50_HPS_FORMAT_FIGEXP M_FIG C_FIG Research in ContextO_ST_ABSWhat is already know about this subject?C_ST_ABS- isomiRs are sequence variants of microRNAs (miRNAs) and may have distinct functional role respect to the canonical sequence. - isomiRs show cell/tissue specificity and are involved in multiple disease contexts. What is the key question?- What is the profile of isomiRs in human pancreatic islets (HI) and in beta cells? - Do isomiRs have a functional role in beta cells? What are the new findings?- IsomiRs represent a relevant fraction of total miRNAs in HI and beta cells. - 3end miRNA sequence modifications are the major fraction of isomiRs in beta cells. - A set of n=11 isomiRs, more expressed than their canonical miRNAs, are enriched in beta cells compared to the other human cell types. - Specific isomiRs are associated with beta cell glucose sensitivity and basal insulin secretion. How might this impact on clinical practice in the foreseeable future?- A comprehensive profile of isomiRs in beta cells may improve our understanding of molecular mechanisms driving beta cell function and dysfunction. - A highly detailed and granular view of miRNAs sequence variants and their expression levels may help in the design of novel therapeutic RNA-based strategies.

molecular biology↗

bioRxiv2021

Increased expression of viral sensor MDA5 in pancreatic islets and in hormone-negative endocrine cells in recent onset type 1 diabetic donors

The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic {beta}-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as Melanoma Differentiation Associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of non-diabetic and T1D donors is still missing. In the present study we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 non-diabetic autoantibody-negative, 2 autoantibody-positive, 8 recent-onset and 2 long-standing T1D). In non-diabetic control donors, MDA5 was expressed both in - and in {beta}-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in -cells. In T1D donors, we observed an increased colocalization rate MDA5-glucagon respect to MDA5-insulin in comparison to non-diabetic controls; such increase was more pronounced in recent onset respect to long standing T1D donors. Of note, an increased colocalization rate MDA5-glucagon was found in insulin-deficient-islets (IDI) respect to insulin containing islets (ICI). Strikingly, in T1D donors we detected the presence of MDA5-positive/hormones-negative endocrine islet-like clusters, putatively deriving from dedifferentiation or neogenesis phoenomena. These clusters were exclusively identified in recent onset donors and not detected in autoantibody-positive non-diabetic or T1D long-standing ones. In conclusion, we showed that MDA5 is preferentially expressed in -cells and its expression is increased in recent onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.

immunology↗

bioRxiv2021

Outer Membrane Vesicles from the gut microbiome contribute to tumor immunity by eliciting cross-reactive T cells

The gut microbiome plays a key role in cancer immunity. One proposed mechanism is through the elicitation of T cells, which incidentally recognize neo-epitopes arising from cancer mutations ("molecular mimicry (MM)" hypothesis). To support MM, Escherichia coli Nissle was engineered with the SIINFEKL epitope (OVA) and orally administered to C57BL/6 mice. The treatment elicited OVA-specific CD8+ T cells in the lamina propria and inhibited the growth of OVA-B16F10 tumors. Importantly, the administration of Outer Membrane Vesicles (OMVs) engineered with different T cell epitopes elicited epitope-specific T cells and inhibited tumor growth. Microbiome shotgun sequencing and TCR sequencing provided evidence that cross-reacting T cells were induced at the mucosal level and subsequently reached the tumor site. Overall, our data support the role of MM in tumor immunity, assign a new role to OMVs and pave the way to new probiotics/OMV-based anti-cancer immunotherapies.

microbiology↗

bioRxiv2021

Reduced miR-184-3p expression occurring in Type 2 diabetic pancreatic islets protects β-cells from lipotoxic and proinflammatory apoptosis via a CRTC1-dependent mechanism

Loss of functional {beta}-cell mass in Type 2 diabetes (T2D) involves molecular mechanisms including {beta}-cell apoptosis, dysfunction, and/or dedifferentiation. MicroRNA miR-184-3p has been demonstrated to be involved in multiple {beta}-cell functions including insulin secretion, proliferation and survival. However, downstream targets and upstream regulators of miR-184-3p have not yet been fully elucidated. Here, we showed that levels of miR-184-3p are reduced in human T2D pancreatic islets and that its reduction protected {beta}-cells from lipotoxic- and inflammatory-induced apoptosis. Interestingly, CREB-Transcriptional Coactivator-1 (CRTC1) is a direct target of miR-184-3p and indeed its expression is upregulated in human T2D pancreatic islets. The downregulation of miR-184-3p in {beta}-cells induced the upregulation of CRTC1 both at mRNA and protein level. Of note, miR-184-3p protection effect was dependent on CRTC1, since its silencing in human {beta}-cells abrogates the protective mechanism exerted by miR-184-3p inhibition. Additionally, we found that the {beta}-cell specific transcription factor NKX6.1, whose DNA binding sites were predicted to be present in human and mouse MIR184 gene promoter sequence, was reduced in T2D human pancreatic islets, in line with miR-184-3p downregulation, and was positively correlated with microRNA expression. Using chromatin immunoprecipitation analysis and mRNA silencing experiments, we demonstrated that NKX6.1 directly controls both human and murine miR-184 expression. In conclusion, we found that miR-184-3p expression is controlled by the {beta}-cell specific transcription factor NKX6.1 and that miR-184-3p reduction protects {beta}-cells from apoptosis through the upregulation of its target gene CRTC1.

cell biology↗

bioRxiv2020

SARS-CoV-2 receptor Angiotensin I-Converting Enzyme type 2 is expressed in human pancreatic islet β-cells and is upregulated by inflammatory stress

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2), is a necessary step for SARS-CoV-2 infection permissiveness. In the light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyse ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing {beta}-cells. ACE2 is also is highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human {beta}-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increases ACE2 expression in the {beta}-cell line EndoC-{beta}H1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct {beta}-cell virus tropism.

cell biology↗