Biology subjects

Cuozzo, F.

Publications and source records attributed to Cuozzo, F..

4 recordsLinked to original sources

bioRxiv2024

Liraglutide treatment reverses unconventional cellular defects in induced pluripotent stem cell-derived β cells harboring a partially functional WFS1 variant

Aims/hypothesisWolfram Syndrome 1 (WS1) is a rare genetic disorder characterized by very heterogeneous clinical manifestations caused by variants of the WFS1 gene, which encodes for the Endoplasmic Reticulum (ER) protein Wolframin, involved in cellular stress response, Ca2+ handling and autophagy. Given the central role of Wolframin, elucidating the impact of WFS1 variants on cell functions is crucial to provide an association with clinical phenotypes. Therefore, as the understanding of patient-specific defects may also help to develop targeted therapeutic approaches, here we aimed at elucidating the impact on {beta} cell function of the c.316-1G>A mutation harboring the partially functional Wolframin that we have previously characterized, and the molecular changes following treatment with the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide. MethodsWe previously generated patient-derived iPSCs (WFS1) and isogenic line in which the c.316-1G>A mutation was genetically corrected (WFS1wt/757A>T), thus performed molecular analysis, including single cell RNAseq (scRNAseq), and functional studies on iPSC-derived {beta} cell (iBeta). Calcium flux imaging and dynamic perifusion assays were used to test glucose responsiveness of iBeta. Treatment with liraglutide was performed to investigate effects on glucose-stimulated insulin secretion (GSIS), unfolded protein response (UPR), autophagy and apoptosis. ResultsWe found that both WFS1 and WFS1wt/757A>T iBeta efficiently differentiated in vitro into pancreatic lineage, but WFS1 showed less mature endocrine phenotype, reduced glucose responsiveness and impaired insulin secretion compared to WFS1wt/757A>T counterpart. The Ca2+ dynamics were altered in WFS1 iBeta as Ca2+ oscillations after glucose challenge were not synchronized mainly due to the CACNA1D and SNAP25 downmodulation. Reduced insulin secretion was correlated with a decrease in PC1/3 levels and overall increase of RGS4 expression in WFS1 iBeta, whereas secretory defects correlated with accelerated autophagic flux. While the functional residual Wolframin in WFS1 iBeta controlled short-term ER stress, prolonged insults or inflammation highlighted ineffective UPR that make the cells unable to escape apoptosis. Interestingly, treatment with liraglutide restored the Ca2+ fluxes and secretory impairments, increasing glucose responsiveness and insulin release of WFS1 iBeta, while protecting these cells from cellular stress and inflammation-induced apoptosis. Conclusion/interpretationOur data highlighted alterations of key cellular pathways involved in WS1 {beta} cell maturation and GSIS and how the pharmacological targeting of the GLP-1/GLP-1R axis was able to restore the physiologic phenotype. This study points out the need to understand the patient-specific molecular determinants associated with WFS1 variants, to design effective therapies to treat the disease.

cell biology↗

bioRxiv2023

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on ER-stress independent β cell apoptosis in a patient with Wolfram syndrome.

Aims/hypothesisWolfram Syndrome 1 (WS1) is an inherited condition mainly manifesting in childhood-onset diabetes mellitus and progressive optic nerve atrophy. The causative gene, WFS1, encodes for Wolframin, a master regulator of several cellular responses, whose mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame ones. As achieving a genotype-phenotype correlation is crucial to deal with disease outcome, works investigating the impact of transcriptional and translational landscapes stemming from such mutations are needed. Therefore, we sought to elucidate the molecular determinants behind the pathophysiological alterations in a WS1 patient carrying compound heterozygous mutations in WFS1 gene: c.316-1G>A, affecting the acceptor splice site (ASS) upstream exon 4, and c.757A>T, introducing a premature termination codon (PTC) in exon 7. MethodsBioinformatic analysis was carried out to infer the alternative splicing events occurring after disruption of ASS, followed by RNAseq and PCR to validate the transcriptional landscape. Patient-derived induced Pluripotent Stem Cells (iPSCs) were used as an in vitro model of WS1 and to investigate the WFS1 alternative splicing isoforms into pancreatic {beta} cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the ER-stress induction and immunotoxicity assays. ResultsWe showed that patient-derived iPSCs retained the ability to differentiate into pancreatic {beta} cells. We demonstrated that the allele carrying the ASS mutation c.316-1G>A originates two PTC-containing alternative splicing transcripts (c.316del and c.316-460del), and two ORF-conserving mRNAs (c.271-513del and c.316-456del) leading to N-terminally truncated polypeptides. By retaining the C-terminal domain, these isoforms sustained the endoplasmic reticulum (ER)-stress response in {beta} cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and pro-inflammatory cytokines affected the NMD-related gene SMG7 expression levels (>2 fold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 {beta} cells, thus resulting in a dramatic accumulation of the PTC-containing isoforms c.316del (>100-fold increase over basal; p<0.001) and c.316-460del (>20-fold increase over basal; p<0.001) and predisposing affected {beta} cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived {beta} cells. Conclusions/interpretationThis study represents a new model to study Wolframin, highlighting how each single mutation of WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 {beta} cells to stress and inflammation, and we postulate that this is triggered by escaping NMD and accumulation of mutated transcripts and truncated proteins. These findings pave the way for further studies on the molecular basis of genotype-phenotype relationship in WS1, to uncover the key determinants that might be targeted to ameliorate the clinical outcome of patients affected by this rare disease.

genetics↗

bioRxiv2022

High-resolution glucose fate-mapping reveals LDHB-dependent lactate production by human pancreatic β cells

Using 13C6 glucose labeling coupled to GC-MS and 2D 1H-13C HSQC NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning {beta} cell function. In both mouse and human islets, the contribution of glucose to the TCA cycle is similar. Pyruvate-fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is six-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and {beta} cell-specific LDHB expression. LDHB inhibition increases glucose-dependent lactate generation in mouse and human {beta} cells, and decreases Ca2+-spiking frequency without affecting ATP/ADP levels. Thus, we show that LDHB limits glucose-stimulated lactate generation in {beta} cells. Further studies are warranted to understand how lactate impacts {beta} cell metabolism and/or function. HIGHLIGHTSO_LIHuman and rodent islets generate lactate following glucose stimulation. C_LIO_LI{beta} cells specifically express LDHB, which acts to limit lactate generation. C_LIO_LILDHB inhibition influences Ca2+ spiking frequency without affecting ATP/ADP ratio. C_LI eTOCCuozzo et al show that glucose-stimulated rodent and human islets generate lactate. Transcriptomic and imaging analyses reveal that LDHB is specifically expressed in {beta} cells and unexpectedly restrains lactate production. LDHB expression and thus regulated lactate generation might reflect a key mechanism underlying {beta} cell metabolism, function and survival.

physiology↗

bioRxiv2020

The prolyl hydroxylase PHD3 maintains β-cell glucose metabolism during fatty acid excess

The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a hypoxia-inducible factor target that uses molecular oxygen to hydroxylate proline. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting {beta}-cells. Here, we show that deletion of PHD3 specifically in {beta}-cells ({beta}PHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, {beta}PHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift away from glycolysis. However, {beta}PHD3KO islets are able to maintain oxidative phosphorylation and insulin secretion by increasing utilization of fatty acids to supply the tricarboxylic acid cycle. This nutrient-sensing switch cannot be sustained and {beta}PHD3KO islets begin to show signs of failure in response to prolonged metabolic stress, including impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the {beta}-cell glucose metabolism machinery by suppressing the use of fatty acids as a primary fuel source, under obesogenic and insulin resistant states. SIGNIFICANCE STATEMENTProlyl-4-hydroxylase 3 (PHD3) is involved in the oxygen-dependent regulation of cell phenotype. A number of recent studies have shown that PHD3 might operate at the interface between oxygen availability and metabolism. To understand how PHD3 influences insulin secretion, which depends on intact glucose metabolism, we generated mice lacking PHD3 specifically in pancreatic {beta}-cells. These mice, termed {beta}PHD3KO, are apparently normal until fed high fat diet at which point their {beta}-cells switch to fatty acids as a fuel source. This switch cannot be tolerated and {beta}-cells in {beta}PHD3KO mice eventually fail. Thus, PHD3 maintains glucose-stimulated insulin secretion in {beta}-cells during states of fatty acid excess, such as diabetes and obesity.

physiology↗