ABSTRACT
OBJECTIVE: The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. METHODS: The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. RESULTS: OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. CONCLUSIONS: Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.
Subject(s)
B-Lymphocytes , Flavin-Adenine Dinucleotide , Flow Cytometry , NAD , Single-Cell Analysis , Humans , Flow Cytometry/methods , NAD/metabolism , Flavin-Adenine Dinucleotide/metabolism , Single-Cell Analysis/methods , B-Lymphocytes/metabolism , Mitochondria/metabolism , T-Lymphocytes/metabolism , Oxidation-Reduction , Fluorescence , Arthritis, Rheumatoid/metabolism , Glycolysis , Oxidative Phosphorylation , Female , Male , Glucose/metabolismABSTRACT
AIMS: Dysregulation of PI3K/Akt/GSK3ß signaling has been implicated in various neurological disorders, including autism spectrum disorder (ASD). G protein-coupled receptor 55 (GPR55) has recently emerged as a potential regulator of this signaling cascade. This study explores the intricate modulation of the PI3K/Akt/GSK3ß signaling cascade via GPR55 activation and its potential therapeutic implications in the context of autism-associated neuronal impairments. MAIN METHODS: Valproic acid (VPA) was administered on embryonic day 12 (E12) to induce ASD, and lysophosphatidylinositol (LPI), a GPR55 agonist, was used prenatally to modulate the receptor activity. Golgi-cox staining was performed to observe neuronal morphology, and Hematoxylin and eosin (H and E) staining was carried out to quantify damaged neurons. Enzyme-linked immunosorbent assay (ELISA) was implemented to identify molecular mediators involved in neuroprotection. KEY FINDINGS: Prenatal VPA exposure resulted in significant abnormalities in synaptic development, which were further evidenced by impairments in social interaction and cognitive function. When LPI was administered, most of the synaptic abnormalities were alleviated, as reflected by higher neuron and dendritic spine count. LPI treatment also reduced cytoplasmic cytochrome c concentration and related neuronal cell death. Mechanistically, GPR55 activation by LPI increases the expression of phospho-Akt and phospho-GSK3ß, leading to the activation of this signaling in the process of rescuing synaptic abnormalities and mitochondria-mediated neuronal apoptosis. SIGNIFICANCE: The observed therapeutic effects of GPR55 activation shed light on its significance as a prospective target for ameliorating mitochondrial dysfunction and dendritic spine loss, offering novel prospects for developing targeted interventions to alleviate the neuropathological causes of ASD.
Subject(s)
Autism Spectrum Disorder , Receptors, G-Protein-Coupled , Humans , Autism Spectrum Disorder/drug therapy , Glycogen Synthase Kinase 3 beta , Lysophospholipids/metabolism , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Receptors, G-Protein-Coupled/metabolism , Valproic Acid/pharmacologyABSTRACT
Pamoic acid is a potent ligand for G protein Coupled Receptor 35 (GPR35) and exhibits antinociceptive property. GPR35 activation leads to increased energy utilization and the expression of anti-inflammatory genes. However, its role in brain disorders, especially in stroke, remains unexplored. Here we show in a mouse model of stroke that GPR35 activation by pamoic acid is neuroprotective. Pharmacological inhibition of GPR35 reveals that pamoic acid reduces infarcts size in a GPR35 dependent manner. The flowcytometric analysis shows the expression of GPR35 on the infiltrating monocytes/macrophages and neutrophils in the ischemic brain. Pamoic acid treatment results in a preferential increment of noninflammatory Ly-6CLo monocytes/macrophages in the ischemic brain along with the reduced neutrophil counts. The neuroprotective effect of GPR35 activation depends on protein kinase B (Akt) and p38 MAPK. Together we conclude that GPR35 activation by pamoic acid reprograms Ly-6CLo monocytes/macrophages to relay a neuroprotective signal into the ischemic brain.
Subject(s)
Brain Ischemia/metabolism , Macrophages/metabolism , Monocytes/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Brain/drug effects , Brain/metabolism , Brain Ischemia/drug therapy , Cerebral Infarction/drug therapy , Cerebral Infarction/metabolism , Disease Models, Animal , Ligands , Macrophages/drug effects , Male , Mice , Monocytes/drug effects , Naphthols/pharmacology , Neuroprotective Agents/pharmacology , Neutrophils/drug effects , Neutrophils/metabolism , Stroke/drug therapy , Stroke/metabolismABSTRACT
BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting a large number of elderly people worldwide. The current therapies for PD are symptom-based; they do not provide a cure but improve the quality of life. Muscular dysfunction is the hallmark clinical feature of PD and oxidative stress and inflammation play a critical role in its pathogenesis. Epalrestat is used for the treatment of diabetic neuropathy and is known to improve antioxidative defense mechanisms in the CNS. Therefore, in this study, we investigated the role of Epalrestat in the reserpine induced mouse model of PD. METHOD: We used Swiss Albino mice for the PD model and tested for akinesia/bradykinesia, muscular rigidity, palpebral ptosis, and tremor, as well as conducting swim and open field tests. Brain samples were used to determine oxidative stress parameters and infiltration of immune cells. RESULTS: Epalrestat treatment significantly improved akinesia and bradykinesia, muscular dysfunctions, tremor level, and gait functions compared to the reserpine group. It also improved the latency in the swim test. Eplarestat significantly reduced lipid peroxidation and NO concentration in different brain tissues and increased the activity of antioxidative enzymes, glutathione, catalase, and superoxide dismutase. Furthermore, Epalrestat reduced neuroinflammation by reducing the number of infiltrating immune cells. CONCLUSION: Eplarestat improves muscular dysfunction in PD by reducing oxidative stress and inflammation.