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Targeting disulfidptosis in cancer: mechanisms and preclinical translation
Project Summary Studying regulated cell death is critical for our understanding of cellular homeostasis and tumor suppression. We recently discovered disulfidptosis as a new form of regulated cell death induced by disulfide stress under NADPH-depleting conditions in SLC7A11-high cancer cells. However, in contrast to our deep understanding of other cell death modalities such as apoptosis and ferroptosis, the molecular and metabolic underpinnings of disulfidptosis, along with its therapeutic implications, remain largely unexplored. The objectives of this application are to elucidate the mechanisms underlying disulfidptosis and to therapeutically target this form of cell death in SLC7A11-high cancers. The proposed studies will make extensive use of human cancer cell lines and integrated human cellbased molecular analyses, including metabolomics, proteomics, CRISPR screening, and biochemical studies, to define the metabolic and signaling mechanisms governing disulfidptosis. In addition, select in vivo studies are incorporated in the therapeutic validation components of the project, where tumor growth response, systemic drug exposure and tolerability, tumor microenvironmental influences, and host immune/stromal interactions must be evaluated in an organismal context to ensure translational rigor. Alternative in vitro systems such as organoids may provide useful complementary information on tumor-intrinsic responses, but they cannot fully recapitulate the systemic metabolic stress, pharmacologic exposure, and organism-level therapeutic efficacy required for these studies. It is expected that our proposed studies will reveal novel mechanisms underlying disulfidptosis and identify effective therapies to induce this form of cell death in SLC7A11-high cancers. Our proposal is highly innovative because it focuses on a previously unexplored cell death pathway in cancer therapy. Our proposed studies will have significant impact on both our understanding of the fundamental mechanisms of disulfidptosis and our ability to target this cell death pathway in cancer treatment.
Metabolic Assessment of Metformin in Pregnancy (MoM-P)
PROJECT SUMMARY The objective of the “Metabolic Assessment of Metformin in Pregnancy “(MoM-P) proposal is to assess the physiological effect of metformin on maternal and neonatal metabolism during pregnancy in individuals developing gestational diabetes (GDM). Metformin is increasingly being used for medical treatment of GDM not adequately treated with nutrition and physical activity. There is inconsistency among various organizations (Society for Maternal Fetal Medicine, American College of Obstetrics and Gynecology and the American Diabetes Association) as to metformin’s role in the medical management of GDM. We will examine the metabolic action of metformin in GDM pregnancies and effect on mothers and their offspring. We plan to recruit 50 participants from Massachusetts General Hospital (MGH) for Specific Aims 1, 2 and 3 and 100 participants from Ohio State University college of Medicine (OSUCOM) for Specific Aims 2 and 3. Participants for the study will have been diagnosed with GDM requiring medical management of GDM as part of the DECIDE multicenter randomized controlled trial. The primary site for DECIDE is OSUCOM, with Dr. Mark Landon as the PI. The MoM-P study will recruit participants from the DECIDE trial at MGH and OSUCOM. The MoM-P study aims are: Aim 1: To establish metformin’s effects on endogenous (primarily hepatic) glucose production (EGP) and insulin sensitivity in late pregnancy. We hypothesize that metformin does not lower EGP in pregnancy and hence the need of additional insulin in the medical management of GDM. We will perform infusion of a stable isotope of glucose (6,6 2H2 glucose) to estimate EGP and a HOMA-IR prior to initiation of medical management and again at 37 weeks gestation. Aim 2: Metformin increases GDF15 levels in human GDM pregnancy and is associated with lower nutrient intake, gestational weight gain (GWG) and increased resting energy expenditure (REE). We hypothesize that metformin increases GDF15 concentrations which lead to GI upset, lower caloric intake/GWG and increases REE. In DECIDE participants randomized to metformin vs. insulin, we will measure GDF15 and examine the relationship to ASA-nutrition records, REE with indirect calorimetry and maternal body composition using air displacement plethysmography (ADP) prior to initiation of medication and again at 37 weeks. Aim 3: To compare fetal growth and body composition in neonates exposed and unexposed to metformin in utero. We hypothesize that metformin treatment of GDM decreases fetal weight: 1) directly based on metformin’s effect on neonatal metabolism (fetal AMPK and mTOR pathways) and 2) indirectly by lowering maternal nutritional intake, fat free mass (FFM) and increasing maternal REE, resulting in decreased neonatal FFM and increased fat mass in childhood. In DECIDE participants, we will measure neonatal body composition with 72 hours of delivery using pediatric ADP and a planned follow-up of children at 2 years in the DECIDE protocol with estimates of male and female children’s body composition.
Versatile treadmill system for measuring locomotion and neural activity in head-fixed mice
Here, we present a protocol for using a versatile treadmill system to measure locomotion and neural activity at high temporal resolution in head-fixed mice. We first describe the assembly of the treadmill system. We then detail surgical implantation of the headplate on the mouse skull, followed by habituation of mice to locomotion on the treadmill system. The system is compact, movable, and simple to synchronize with other data streams, making it ideal for monitoring brain activity in diverse behavioral frameworks. https://dx.doi.org/10.1016/j.xpro.2022.101701
Metabolic spikes: from rogue electrons to Parkinson's
Conventionally, neurons are thought to be cellular units that process synaptic inputs into synaptic spikes. However, it is well known that neurons can also spike spontaneously and display a rich repertoire of firing properties with no apparent functional relevance e.g. in in vitro cortical slice preparations. In this talk, I will propose a hypothesis according to which intrinsic excitability in neurons may be a survival mechanism to minimize toxic byproducts of the cell’s energy metabolism. In neurons, this toxicity can arise when mitochondrial ATP production stalls due to limited ADP. Under these conditions, electrons deviate from the electron transport chain to produce reactive oxygen species, disrupting many cellular processes and challenging cell survival. To mitigate this, neurons may engage in ADP-producing metabolic spikes. I will explore the validity of this hypothesis using computational models that illustrate the implications of synaptic and metabolic spiking, especially in the context of substantia nigra pars compacta dopaminergic neurons and their degeneration in Parkinson's disease.
Parp mutations protect from mitochondrial toxicity in Alzheimer’s disease
Alzheimer’s disease is the most common age-related neurodegenerative disorder. Familial forms of Alzheimer’s disease associated with the accumulation of a toxic form of amyloid-β (Aβ) peptides are linked to mitochondrial impairment. The coenzyme nicotinamide adenine dinucleotide (NAD+) is essential for both mitochondrial bioenergetics and nuclear DNA repair through NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Here, we analysed the metabolomic changes in flies over-expressing Aβ and showed a decrease of metabolites associated with nicotinate and nicotinamide metabolism, which is critical for mitochondrial function in neurons. We show that increasing the bioavailability of NAD+ protects against Aβ toxicity. Pharmacological supplementation using NAM, a form of vitamin B that acts as a precursor for NAD+ or a genetic mutation of PARP rescues mitochondrial defects, protects neurons against degeneration and reduces behavioural impairments in a fly model of Alzheimer’s disease. Next, we looked at links between PARP polymorphisms and vitamin B intake in patients with Alzheimer’s disease. We show that polymorphisms in the human PARP1 gene or the intake of vitamin B, are associated with a decrease in the risk and severity of Alzheimer’s disease. We suggest that enhancing the availability of NAD+ by either vitamin B supplements or the inhibition of NAD+-dependent enzymes, such as PARPs are potential therapies for Alzheimer’s disease.
Carnosine negatively modulates pro-oxidant activities of M1 peripheral macrophages and prevents neuroinflammation induced by amyloid-β in microglial cells
Carnosine is a natural dipeptide widely distributed in mammalian tissues and exists at particularly high concentrations in skeletal and cardiac muscles and brain. A growing body of evidence shows that carnosine is involved in many cellular defense mechanisms against oxidative stress, including inhibition of amyloid-β (Aβ) aggregation, modulation of nitric oxide (NO) metabolism, and scavenging both reactive nitrogen and oxygen species. Different types of cells are involved in the innate immune response, with macrophage cells representing those primarily activated, especially under different diseases characterized by oxidative stress and systemic inflammation such as depression and cardiovascular disorders. Microglia, the tissue-resident macrophages of the brain, are emerging as a central player in regulating key pathways in central nervous system inflammation; with specific regard to Alzheimer’s disease (AD) these cells exert a dual role: on one hand promoting the clearance of Aβ via phagocytosis, on the other hand increasing neuroinflammation through the secretion of inflammatory mediators and free radicals. The activity of carnosine was tested in an in vitro model of macrophage activation (M1) (RAW 264.7 cells stimulated with LPS + IFN-γ) and in a well-validated model of Aβ-induced neuroinflammation (BV-2 microglia treated with Aβ oligomers). An ample set of techniques/assays including MTT assay, trypan blue exclusion test, high performance liquid chromatography, high-throughput real-time PCR, western blot, atomic force microscopy, microchip electrophoresis coupled to laser-induced fluorescence, and ELISA aimed to evaluate the antioxidant and anti-inflammatory activities of carnosine was employed. In our experimental model of macrophage activation (M1), therapeutic concentrations of carnosine exerted the following effects: 1) an increased degradation rate of NO into its non-toxic end-products nitrite and nitrate; 2) the amelioration of the macrophage energy state, by restoring nucleoside triphosphates and counterbalancing the changes in ATP/ADP, NAD+/NADH and NADP+/NADPH ratio obtained by LPS + IFN-γ induction; 3) a reduced expression of pro-oxidant enzymes (NADPH oxidase, Cyclooxygenase-2) and of the lipid peroxidation product malondialdehyde; 4) the rescue of antioxidant enzymes expression (Glutathione peroxidase 1, Superoxide dismutase 2, Catalase); 5) an increased synthesis of transforming growth factor-β1 (TGF-β1) combined with the negative modulation of interleukines 1β and 6 (IL-1β and IL-6), and 6) the induction of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1). In our experimental model of Aβ-induced neuroinflammation, carnosine: 1) prevented cell death in BV-2 cells challenged with Aβ oligomers; 2) lowered oxidative stress by decreasing the expression of inducible nitric oxide synthase and NADPH oxidase, and the concentrations of nitric oxide and superoxide anion; 3) decreased the secretion of pro-inflammatory cytokines such as IL-1β simultaneously rescuing IL-10 levels and increasing the expression and the release of TGF-β1; 4) prevented Aβ-induced neurodegeneration in primary mixed neuronal cultures challenged with Aβ oligomers and these neuroprotective effects was completely abolished by SB431542, a selective inhibitor of type-1 TGF-β receptor. Overall, our data suggest a novel multimodal mechanism of action of carnosine underlying its protective effects in macrophages and microglia and the therapeutic potential of this dipeptide in counteracting pro-oxidant and pro-inflammatory phenomena observed in different disorders characterized by elevated levels of oxidative stress and inflammation such as depression, cardiovascular disorders, and Alzheimer’s disease.
On the purpose and origin of spontaneous neural activity
Spontaneous firing, observed in many neurons, is often attributed to ion channel or network level noise. Cortical cells during slow wave sleep exhibit transitions between so called Up and Down states. In this sleep state, with limited sensory stimuli, neurons fire in the Up state. Spontaneous firing is also observed in slices of cholinergic interneurons, cerebellar Purkinje cells and even brainstem inspiratory neurons. In such in vitro preparations, where the functional relevance is long lost, neurons continue to display a rich repertoire of firing properties. It is perplexing that these neurons, instead of saving their energy during information downtime and functional irrelevance, are eager to fire. We propose that spontaneous firing is not a chance event but instead, a vital activity for the well-being of a neuron. We postulate that neurons, in anticipation of synaptic inputs, keep their ATP levels at maximum. As recovery from inputs requires most of the energy resources, neurons are ATP surplus and ADP scarce during synaptic quiescence. With ADP as the rate-limiting step, ATP production stalls in the mitochondria when ADP is low. This leads to toxic Reactive Oxygen Species (ROS) formation, which are known to disrupt many cellular processes. We hypothesize that spontaneous firing occurs at these conditions - as a release valve to spend energy and to restore ATP production, shielding the neuron against ROS. By linking a mitochondrial metabolism model to a conductance-based neuron model, we show that spontaneous firing depends on baseline ATP usage and on ATP-cost-per-spike. From our model, emerges a mitochondrial mediated homeostatic mechanism that provides a recipe for different firing patterns. Our findings, though mostly affecting intracellular dynamics, may have large knock-on effects on the nature of neural coding. Hitherto it has been thought that the neural code is optimised for energy minimisation, but this may be true only when neurons do not experience synaptic quiescence.
Brainstem control of a state-dependent motor response reversal in Xenopus laevis tadpoles
FENS Forum 2024
The developmental effects of repeated antenatal dexamethasone treatment on ADP-mediated and adenosinergic signaling system in the auditory brainstem of C57BL/6 mice
FENS Forum 2024
Identification of bilateral homeostatic plasticity in olfactory glomeruli of X. tropicalis tadpoles
FENS Forum 2024
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