Drug-Induced Lysosomal Impairment Is Associated with the Release of Extracellular Vesicles Carrying Autophagy Markers

Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal–lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities.

Download Full-text

Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

International Journal of Molecular Sciences ◽

10.3390/ijms22094637 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4637

Author(s):

Daniel Barth ◽

Andreas Lückhoff ◽

Frank J. P. Kühn

Keyword(s):

Amino Acid Residues ◽

Hek 293 ◽

Complete Inactivation ◽

293 Cells ◽

Activation Mechanisms ◽

Specific Regulation ◽

Species Specific ◽

Inside Out ◽

Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

Download Full-text

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Download Full-text

Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004349 ◽

2018 ◽

Vol 293 (44) ◽

pp. 16984-16993 ◽

Cited By ~ 6

Author(s):

Antje Cordshagen ◽

Wiebke Busch ◽

Michael Winklhofer ◽

Hans Gerd Nothwang ◽

Anna-Maria Hartmann

Keyword(s):

Intrinsic Activity ◽

Hek 293 ◽

Flexible Control ◽

Mediated Effects ◽

293 Cells ◽

Bona Fide ◽

Graded Activity ◽

First Time ◽

Increased Activity

The pivotal role of K+-Cl− cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

Download Full-text

Anoctamin 5/TMEM16E facilitates muscle precursor cell fusion

The Journal of General Physiology ◽

10.1085/jgp.201812097 ◽

2018 ◽

Vol 150 (11) ◽

pp. 1498-1509 ◽

Cited By ~ 14

Author(s):

Jarred M. Whitlock ◽

Kuai Yu ◽

Yuan Yuan Cui ◽

H. Criss Hartzell

Keyword(s):

Cell Fusion ◽

Ionic Current ◽

Ionic Currents ◽

Muscle Physiology ◽

Muscle Repair ◽

Hek 293 ◽

293 Cells ◽

Phospholipid Scrambling ◽

Muscle Precursor Cell

Limb-girdle muscular dystrophy type 2L (LGMD2L) is a myopathy arising from mutations in ANO5; however, information about the contribution of ANO5 to muscle physiology is lacking. To explain the role of ANO5 in LGMD2L, we previously hypothesized that ANO5-mediated phospholipid scrambling facilitates cell–cell fusion of mononucleated muscle progenitor cells (MPCs), which is required for muscle repair. Here, we show that heterologous overexpression of ANO5 confers Ca2+-dependent phospholipid scrambling to HEK-293 cells and that scrambling is associated with the simultaneous development of a nonselective ionic current. MPCs isolated from adult Ano5−/− mice exhibit defective cell fusion in culture and produce muscle fibers with significantly fewer nuclei compared with controls. This defective fusion is associated with a decrease of Ca2+-dependent phosphatidylserine exposure on the surface of Ano5−/− MPCs and a decrease in the amplitude of Ca2+-dependent outwardly rectifying ionic currents. Viral introduction of ANO5 in Ano5−/− MPCs restores MPC fusion competence, ANO5-dependent phospholipid scrambling, and Ca2+-dependent outwardly rectifying ionic currents. ANO5-rescued MPCs produce myotubes having numbers of nuclei similar to wild-type controls. These data suggest that ANO5-mediated phospholipid scrambling or ionic currents play an important role in muscle repair.

Download Full-text

Extracellular vesicles, stem cells and the role of miRNAs in neurodegeneration

Current Neuropharmacology ◽

10.2174/1570159x19666210817150141 ◽

2021 ◽

Vol 19 ◽

Author(s):

Ayaz M. Belkozhayev ◽

Minnatallah Al-Yozbaki ◽

Alex George ◽

Raigul Ye Niyazova ◽

Kamalidin O. Sharipov ◽

...

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Extracellular Vesicles ◽

Intercellular Communication ◽

Direct Consequence ◽

Therapeutic Benefit ◽

The Body ◽

A Cell ◽

Crucial Information

There are different modalities of intercellular communication governed by cellular homeostasis. In this review, we will explore one of these forms of communication called extracellular vesicles (EVs). These vesicles are released by all cells in the body and are heterogeneous in nature. The primary function of EVs is to share information through their cargo consisting of proteins, lipids and nucleic acids (mRNA, miRNA, dsDNA etc.) with other cells, which have a direct consequence on their microenvironment. We will focus on the role of EVs of mesenchymal stem cells (MSCs) in the nervous system and how these participate in intercellular communication to maintain physiological function and provide neuroprotection. However, deregulation of this same communication system could play a role in several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, multiple sclerosis, prion disease and Huntington’s disease. The release of EVs from a cell provides crucial information to what is happening inside the cell and thus could be used in diagnostics and therapy. We will discuss and explore new avenues for the clinical applications of using engineered MSC-EVs and their potential therapeutic benefit in treating neurodegenerative diseases.

Download Full-text

The Role of Extracellular Vesicles in Cutaneous Remodeling and Hair Follicle Dynamics

International Journal of Molecular Sciences ◽

10.3390/ijms20112758 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2758 ◽

Cited By ~ 8

Author(s):

Elisa Carrasco ◽

Gonzalo Soto-Heredero ◽

María Mittelbrunn

Keyword(s):

Hair Follicle ◽

Extracellular Vesicles ◽

Cell Function ◽

Hair Follicles ◽

The Body ◽

Epithelial Stem Cells ◽

Therapeutic Approaches ◽

Waste Products ◽

Efficient Treatment

Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are cell-derived membranous structures that were originally catalogued as a way of releasing cellular waste products. Since the discovery of their function in intercellular communication as carriers of proteins, lipids, and DNA and RNA molecules, numerous therapeutic approaches have focused on the use of EVs, in part because of their minimized risk compared to cell-based therapies. The skin is the organ with the largest surface in the body. Besides the importance of its body barrier function, much attention has been paid to the skin in regenerative medicine because of its cosmetic aspect, which is closely related to disorders affecting pigmentation and the presence or absence of hair follicles. The use of exosomes in therapeutic approaches for cutaneous wound healing has been reported and is briefly reviewed here. However, less attention has been paid to emerging interest in the potential capacity of EVs as modulators of hair follicle dynamics. Hair follicles are skin appendices that mainly comprise an epidermal and a mesenchymal component, with the former including a major reservoir of epithelial stem cells but also melanocytes and other cell types. Hair follicles continuously cycle, undergoing consecutive phases of resting, growing, and regression. Many biomolecules carried by EVs have been involved in the control of the hair follicle cycle and stem cell function. Thus, investigating the role of either naturally produced or therapeutically delivered EVs as signaling vehicles potentially involved in skin homeostasis and hair cycling may be an important step in the attempt to design future strategies towards the efficient treatment of several skin disorders.

Download Full-text

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00288.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. F1028-F1034 ◽

Cited By ~ 26

Author(s):

W. Bruce Sneddon ◽

Yanmei Yang ◽

Jianming Ba ◽

Lisa M. Harinstein ◽

Peter A. Friedman

Keyword(s):

Conditioned Media ◽

Kidney Cells ◽

Wild Type ◽

Egfr Transactivation ◽

Hek 293 ◽

Erk Activation ◽

Hek 293 Cells ◽

293 Cells ◽

Extracellular Signal

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

Download Full-text

The Protective Effect of Exercise in Neurodegenerative Diseases: The Potential Role of Extracellular Vesicles

Cells ◽

10.3390/cells9102182 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2182 ◽

Cited By ~ 1

Author(s):

Oliver K Fuller ◽

Martin Whitham ◽

Suresh Mathivanan ◽

Mark A Febbraio

Keyword(s):

Physical Activity ◽

Extracellular Vesicles ◽

Potential Role ◽

The Body ◽

Therapeutic Effects ◽

Health And Wellbeing ◽

Systemic Factors ◽

Diabetes And Obesity

Physical activity has systemic effects on the body, affecting almost every organ. It is important not only for general health and wellbeing, but also in the prevention of diseases. The mechanisms behind the therapeutic effects of physical activity are not completely understood; however, studies indicate these benefits are not confined to simply managing energy balance and body weight. They also include systemic factors which are released into the circulation during exercise and which appear to underlie the myriad of benefits exercise can elicit. It was shown that along with a number of classical cytokines, active tissues also engage in inter-tissue communication via extracellular vesicles (EVs), specifically exosomes and other small EVs, which are able to deliver biomolecules to cells and alter their metabolism. Thus, EVs may play a role in the acute and systemic adaptations that take place during and after physical activity, and may be therapeutically useful in the treatment of a range of diseases, including metabolic disorders such as type 2 diabetes and obesity; and the focus of this review, neurological disorders such as Alzheimer’s disease.

Download Full-text