We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn(2+) binding, have affinities that span the pico- to ...nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn(2+) was buffered at 0.4 nM in pancreatic beta cells, and we found substantially higher Zn(2+) concentrations in insulin-containing secretory vesicles.
Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca(2+) influx. As well as activating ATP-consuming processes, cytosolic Ca(2+) ...increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca(2+) transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca(2+) uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca(2+) and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca(2+) uniporter, MCU, is required for depolarisation-induced mitochondrial Ca(2+) increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na(+)-Ca(2+) exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca(2+) uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca(2+) accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.
Several observational studies investigated risk factors for suicide attempts/completed suicides in older age with contrasting evidence from ongoing population-based research. The risk factors most ...associated to suicide attempts than other variables were: depressive disorders, methods employed to self-harm (particularly poisoning), and psychotropic drug utilization followed by psychological factors and disability. Moreover, male sex, violent methods to self-harm, any psychiatric disorder (depression, anxiety and bipolar disorders), a poor medical condition, stressors/bereavement, and living alone appeared to be more significant for predicting completed suicides in late life. There is growing evidence of a role of environmental exposures in the pathogenesis and epigenetics of suicidal behavior in older age. Little is known about the possible relationship between suicidal ideation in older age and its biopsychosocial predictors, although psychiatric disorders (among which late-life depression, LLD), play a fundamental role. LLD, distinguished as late-onset depression (LOD) and early-onset depression (EOD). Suicidal ideators accounted for 2.32% of subjects, were female, smokers and obese affected by multimorbidity. After adjusting for age, gender, education and social dysfunction, suicidal ideation was associated to LLD (EOD>LOD:OR:21.71, 95% CI:9.22-51.14). In the full random forest model, asthma was the most important contributor to suicidal ideation. Among biomarkers, interleukin (IL)-6 followed by tumor necrosis factor (TNF)-a, Apolipoprotein E e4 allele-carriers, C-reactive protein contributed most to suicidal ideation. Although EOD is a strong determinant of suicidal ideation, other non-psychiatric factors, i.e., serum inflammation biomarkers, APOE e4 allele, and multimorbidity, should be taken into account when evaluating a suicidal ideation phenotype in older age.
Disclosure
No significant relationships.
Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum ...(ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration (ATPCYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration (Ca(2+)CYT), and additionally, diminished ATPCYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis.
Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in ...three dimensions. Exploiting the fact that insulin granules contain a high level of Zn2+, and that Zn2+ is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn2+ chelation and bound Zn2+ with high selectivity against Ca2+ and Mg2+. When added to cultured β cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn2+ concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled β cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn2+/insulin release occurred largely in small groups of adjacent β cells, with each forming a "secretory unit." Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca2+ elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca2+ signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn2+-containing granules, ZIMIR may find applications in β-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.
The modern concept of stress is based on responses to events or factors ("stressors") experienced as aversive, threatening or excessive for maintaining physiological equilibrium of an organism. ...Prolonged exposure to stressors, particularly during early life, is strongly associated with later psychiatric disorders. Underlying mechanistic connections between stress responses and development of psychiatric illnesses remain uncertain and typically appear to be nonspecific. Relevant candidate mechanisms are likely to include the hypothalamic-pituitary-adrenal (HPA) axis, marked by sustained excessive release of cortisol from the adrenal cortex. In turn, this process is influenced by and alters various central neurotransmitter and other molecular signaling systems that include glutamate, dopamine, serotonin, and neurotrophic peptides. Additional manifestations of stress include altered neurogenesis and neuroplasticity, as well as oxidative neuron-damaging effects. The complex molecular systems involved in these processes present many opportunities for innovative pharmacological interventions that may have preventive or therapeutic benefits regarding mental illnesses arising from stress.
Introduction
Major depressive disorder (MDD), especially in case of suicidal risk, is a psychiatric emergency, associated with high patient burden and healthcare resource utilization. Although active ...and urgent treatment is crucial, little is known on comprehensive care management of this condition in Italy.
Objectives
Here we report the ARIANNA study NCT04463108 interim results to primarily describe the treatment utilization pathways of patients with MDD and active suicidal ideation with intent in the current clinical practice in Italy.
Methods
This observational prospective cohort study included adult patients with a moderate-to-severe major depressive episode (MDE) and active suicidality from 24 Italian sites. Real-world data on patient characteristics, treatments, clinical outcomes, and healthcare utilization were collected during a 90-day follow-up. Data collection is still ongoing.
Results
Sixty-four evaluable patients were considered for this interim analysis: 41 (64.1%) females, mean SD age 46.0 15.4 years, a concomitant psychiatric diagnosis in 7 (10.9%), and other comorbidities in 26 (40.6%). The baseline mean SD MADRS total score was 37.5 7.2, with severe MDE and prior suicidal behavior in 30 (46.9%) and 21 (32.8%) patients, respectively. Median 25th;75th percentiles duration of current MDE was 1.1 0.3;2.1 months. Acute inpatient hospitalization was provided for 43 (67.2%) patients. Antidepressant augmentation with mood stabilizers and/or antipsychotic drugs and optimization were the most frequent early standard-of-care treatment regimens in 32 (53.3%) and 24 (40.0%) patients with available data (N=60), respectively.
Conclusions
Our preliminary results suggest that initial treatment approaches in this critical population are mostly polypharmacological and delivered as inpatient care, with consequent intensive resource utilization.
Disclosure
The ARIANNA study was sponsored by Janssen-Cilag SpA, Italy. DD and MA are employees of Janssen-Cilag SpA. DA and BR are employees of MediNeos S.U.R.L., a company subject to the direction and coordination of IQVIA Solutions HQ Ltd.
Zinc (Zn2+) ions are increasingly recognized as playing an important role in cellular physiology. Whereas the free Zn2+ concentration in the cytosol has been established to be 0.1–1 nM, the free Zn2+ ...concentration in subcellular organelles is not well-established. Here, we extend the eCALWY family of genetically encoded Förster Resonance Energy Transfer (FRET) Zn2+ probes to permit measurements in the endo(sarco)plasmic reticulum (ER) and mitochondrial matrix. Deployed in a variety of mammalian cell types, these probes reveal resting mitochondrial free Zn2+ values of ∼300 pM, somewhat lower than in the cytosol but 3 orders of magnitude higher than recently reported using an alternative FRET-based sensor. By contrast, free ER Zn2+ was found to be ≥5 nM, which is >5000-fold higher than recently reported but consistent with the proposed role of the ER as a mobilizable Zn2+ store. Treatment of β-cells or cardiomyocytes with sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors, mobilization of ER Ca2+ after purinergic stimulation with ATP, or manipulation of ER redox, exerted no detectable effects on Zn2+ER. These findings question the previously proposed role of Ca2+ in Zn2+ mobilization from the ER and suggest that high ER Zn2+ levels may be an important aspect of cellular homeostasis.
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