α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate
. J Biol Chem 2020;Abstract
α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI4,5P) and phosphatidylinositol 3,4-bisphosphate (PI3,4P). Our results show that α-Syn colocalizes with PIP and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI4,5P levels on the plasma membrane. In accord with their effects on PI4,5P levels, the PD associated A30P, E46K and A53T mutations in α-Syn further enhance CME in neuronal and non-neuronal cells. However, Lysine to Glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, that interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI4,5P levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn-mediated membrane trafficking.
Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells' heterogeneity
. Nat Commun 2020;11(1):4516.Abstract
Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.
Aripiprazole Cytotoxicity Coincides with Activation of the Unfolded Protein Response in Human Hepatic Cells
. J Pharmacol Exp Ther 2020;374(3):452-461.Abstract
Schizophrenia is a mental disease that results in decreased life expectancy and well-being by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. Although the second-generation antipsychotics (SGA), Olanzapine and Aripiprazole, are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain, and dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We therefore investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma, and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of inositol-requiring enzyme 1 (IRE1)and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), two major transducers of the UPR. Cells underwent apoptosis with Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2, a human liver cancer cell line, protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress. SIGNIFICANCE STATEMENT: The antischizophrenic drug Aripiprazole exerts cytotoxic properties at high concentrations. This study shows that this cytotoxicity is associated with the induction of endoplasmic reticulum (ER) stress and IRE1 activation, mechanisms involved in diet-induced obesity. Aripiprazole induced ER stress and calcium mobilization from the ER in human and mouse hepatocytes. Our study highlights a new mechanism of Aripiprazole that is not related to its effect on dopamine signaling.
Dual-Targeted Autoimmune Sword in Fatal Epilepsy: Patient's glutamate receptor AMPA GluR3B peptide autoimmune antibodies bind, induce Reactive Oxygen Species (ROS) in, and kill both human neural cells and T cells
. J Autoimmun 2020;112:102462.Abstract
Nodding Syndrome (NS) is a fatal pediatric epilepsy of unknown etiology, accompanied by multiple neurological impairments, and associated with Onchocerca volvulus (Ov), malnutrition, war-induced trauma, and other insults. NS patients have neuroinflammation, and ~50% have cross-reactive Ov/Leiomodin-1 neurotoxic autoimmune antibodies. RESULTS: Studying 30 South Sudanese NS patients and a similar number of healthy subjects from the same geographical region, revealed autoimmune antibodies to 3 extracellular peptides of ionotropic glutamate receptors in NS patients: AMPA-GluR3B peptide antibodies (86%), NMDA-NR1 peptide antibodies (77%) and NMDA-NR2 peptide antibodies (87%) (in either 1:10, 1:100 or 1:1000 serum dilution). In contrast, NS patients did not have 26 other well-known autoantibodies that target the nervous system in several autoimmune-mediated neurological diseases. We demonstrated high expression of both AMPA-GluR3 and NMDA-NR1 in human neural cells, and also in normal human CD3 T cells of both helper CD4 and cytotoxic CD8 types. Patient's GluR3B peptide antibodies were affinity-purified, and by themselves precipitated short 70 kDa neuronal GluR3. NS patient's affinity-purified GluR3B peptide antibodies also bound to, induced Reactive Oxygen Species (ROS) in, and killed both human neural cells and T cells within 1-2 hours only. NS patient's purified IgGs, or serum (1:10 or 1:30), induced similar effects. In vivo video EEG experiments in normal mice, revealed that when NS patient's purified IgGs were released continuously (24/7 for 1 week) in normal mouse brain, they induced all the following: 1.Seizures, 2. Cerebellar Purkinje cell loss, 3. Degeneration in the hippocampus and cerebral cortex, and 4. Elevation of CD3 T cells, and of activated Mac-2microglia and GFAPastrocytes in both the gray and white matter of the cerebral cortex, hippocampus, corpus calossum and cerebellum of mice. NS patient's serum cytokines: IL-1β, IL-2, IL-6, IL-8, TNFα, IFNγ, are reduced by 85-99% compared to healthy subjects, suggesting severe immunodeficiency in NS patients. This suspected immunodeficiency could be caused by combined effects of the: 1. Chronic Ov infection, 2. Malnutrition, 3. Killing of NS patient's T cells by patient's own GluR3B peptide autoimmune antibodies (alike the killing of normal human T cells by the NS patient's GluR3B peptide antibodies found herein in vitro). CONCLUSIONS: Regardless of NS etiology, NS patients suffer from 'Dual-targeted Autoimmune Sword': autoimmune AMPA GluR3B peptide antibodies that bind, induce ROS in, and kill both neural cells and T cells. These neurotoxic and immunotoxic GluR3B peptide autoimmune antibodies, and also NS patient's NMDA-NR1/NR2A and Ov/Leiomodin-1 autoimmune antibodies, must be silenced or removed. Moreover, the findings of this study are relevant not only to NS, but also to many more patients with other types of epilepsy, which have GluR3B peptide antibodies in serum and/or CSF. This claim is based on the following facts: 1. The GluR3 subunit is expressed in neural cells in crucial brains regions, in motor neurons in the spinal cord, and also in other cells in the body, among them T cells of the immune system, 2. The GluR3 subunit has diverse neurophysiological role, and its deletion or abnormal function can: disrupt oscillatory networks of both sleep and breathing, impair motor coordination and exploratory activity, and increase the susceptibility to generate seizures, 3. GluR3B peptide antibodies were found so far in ~27% of >300 epilepsy patients worldwide, which suffer from various other types of severe, intractable and enigmatic epilepsy, and which turned out to be 'Autoimmune Epilepsy'. Furthermore, the findings of this study could be relevant to different neurological diseases besides epilepsy, since other neurotransmitter-receptors autoantibodies are present in other neurological and psychiatric diseases, e.g. autoimmune antibodies against other GluRs, Dopamine receptors, GABA receptors, Acetylcholine receptors and others. These neurotransmitter-receptors autoimmune autoantibodies might also act as 'Dual-targeted Autoimmune Sword' and damage both neural cells and T cells (as the AMPA-GluR3B peptide antibodies induced in the present study), since T cells, alike neural cells, express most if not all these neurotransmitter receptors, and respond functionally to the respective neurotransmitters - a scientific and clinical topic we coined 'Nerve-Driven Immunity'.
Anandamide prevents the adhesion of filamentous Candida albicans to cervical epithelial cells
. Sci Rep 2020;10(1):13728.Abstract
Candidiasis is a fungal infection caused by Candida species that have formed a biofilm on epithelial linings of the body. The most frequently affected areas include the vagina, oral cavity and the intestine. In severe cases, the fungi penetrate the epithelium and cause systemic infections. One approach to combat candidiasis is to prevent the adhesion of the fungal hyphae to the epithelium. Here we demonstrate that the endocannabinoid anandamide (AEA) and the endocannabinoid-like N-arachidonoyl serine (AraS) strongly prevent the adherence of C. albicans hyphae to cervical epithelial cells, while the endocannabinoid 2-arachidonoylglycerol (2-AG) has only a minor inhibitory effect. In addition, we observed that both AEA and AraS prevent the yeast-hypha transition and perturb hyphal growth. Real-time PCR analysis showed that AEA represses the expression of the HWP1 and ALS3 adhesins involved in Candida adhesion to epithelial cells and the HGC1, RAS1, EFG1 and ZAP1 regulators of hyphal morphogenesis and cell adherence. On the other hand, AEA increased the expression of NRG1, a transcriptional repressor of filamentous growth. Altogether, our data show that AEA and AraS have potential anti-fungal activities by inhibiting hyphal growth and preventing hyphal adherence to epithelial cells.
Bezafibrate Improves Mitochondrial Fission and Function in DNM1L-Deficient Patient Cells
. Cells 2020;9(2)Abstract
Mitochondria are involved in many cellular processes and their main role is cellular energy production. They constantly undergo fission and fusion, and these counteracting processes are under strict balance. The cytosolic dynamin-related protein 1, Drp1, or dynamin-1-like protein (DNM1L) mediates mitochondrial and peroxisomal division. Defects in the gene result in a complex neurodevelopmental disorder with heterogeneous symptoms affecting multiple organ systems. Currently there is no curative treatment available for this condition. We have previously described a patient with a de novo heterozygous c.1084G>A (p.G362S) mutation and studied the effects of a small molecule, bezafibrate, on mitochondrial functions in this patient's fibroblasts compared to controls. Bezafibrate normalized growth on glucose-free medium, as well as ATP production and oxygen consumption. It improved mitochondrial morphology in the patient's fibroblasts, although causing a mild increase in ROS production at the same time. A human foreskin fibroblast cell line overexpressing the p.G362S mutation showed aberrant mitochondrial morphology, which normalized in the presence of bezafibrate. Further studies would be needed to show the consistency of the response to bezafibrate, possibly using fibroblasts from patients with different mutations in , and this treatment should be confirmed in clinical trials. However, taking into account the favorable effects in our study, we suggest that bezafibrate could be offered as a treatment option for patients with certain mutations.