The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization

Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

doi:10.1101/pdb.prot087106

Cited by 30 publications

(23 citation statements)

References 4 publications

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“…NAC also maintained lysosomal pH in rPMs exposed to cART. LMP is the major cause of proton leakage through a destabilized membrane, resulting in loss of the pH gradient (65,66). Changes in LMP were further confirmed by assessing the GAL3 translocation into the damaged lysosomes.…”

Section: Discussionmentioning

confidence: 86%

N-Acetylcysteine Reverses Antiretroviral-Mediated Microglial Activation by Attenuating Autophagy-Lysosomal Dysfunction

et al. 2020

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Successful suppression of viral replication by combined antiretroviral therapy (cART) in HIV-1 infected individuals is paradoxically also accompanied by an increased prevalence of HIV-associated neurocognitive disorders (HAND) in these individuals. HAND is characterized by a state of chronic oxidative stress and inflammation. Microglia are extremely sensitive to a plethora of stimuli, including viral proteins and cART. The current study aimed to assess the effects of cART-mediated oxidative stress on the induction of inflammatory responses in microglia. In the present study, we chose a combination of three commonly used antiretroviral drugs-tenofovir disoproxil fumarate, emtricitabine, and dolutegravir. We demonstrated that exposure of microglia to the chosen cART cocktail induced generation of reactive oxygen species, subsequently leading to lysosomal dysfunction and dysregulated autophagy, ultimately resulting in the activation of microglia. Intriguingly, the potent antioxidant, N-acetylcysteine, reversed the damaging effects of cART. These in vitro findings were further corroborated in vivo wherein cART-treated HIV transgenic (Tg) rats demonstrated increased microglial activation, exaggerated lysosome impairment, and dysregulated autophagy in the prefrontal cortices compared with HIV Tg rats not exposed to cART. Similar to in vitro findings, the treatment of HIV Tg rats with N-acetylcysteine also mitigated the deleterious effects of cART. Taken together, our findings suggest that oxidative stress-mediated lysosomal dysfunction plays a critical role in the pathogenesis of HAND in drug-treated HIV-infected individuals and that antioxidant-mediated mitigation of oxidative stress could thus be considered as an adjunctive therapeutic strategy for ameliorating/dampening some of the neurological complications of HAND.

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Section: Discussionmentioning

confidence: 86%

N-Acetylcysteine Reverses Antiretroviral-Mediated Microglial Activation by Attenuating Autophagy-Lysosomal Dysfunction

et al. 2020

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“…Next, we stained cells with Acridine Orange, which accumulates in compartments with low pH, to determine the concentration of LLOMe that causes lysosomal membranes to lose the proton gradient (Repnik et al, 2016c). As early as 10 min after treatment, the proton gradient of acidic vesicles was lost at concentrations as low as 0.25 mM LLOMe (Fig.…”

Section: Apoptosis Is Induced In Hela Cells By Concentrations Of Llommentioning

confidence: 99%

“…The assay was as described previously (Repnik et al, 2016c). For microscopic analysis, cells were seeded in four-chamber glass-bottom wells (Lab-Tec, Nunc).…”

Section: Staining Lysosomes With Weak Basic Dyesmentioning

confidence: 99%

L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes

Repnik

Distefano

Speth

et al. 2017

Journal of Cell Science

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L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis.

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“…To further determine the biochemical features of this type of cell death in PPV-infected cells, we used lysosome tracker DND-26 and lysosomal proteases (cathepsin D and L) to monitor lysosomal membrane permeabilization of PPV-infected cells. Lysosome tracker DND-26 is accumulated in acidic organelles in live cells and released from lysosomes when lysosomal membrane permeabilization increases or lysosomes are damaged [37]. Compared with mock infection, PPV infection markedly decreased the mean fluorescence intensity (MFI) of DND-26 ( Figure 4D).…”

Section: Ppv Can Induce Non-apoptotic Death In Ptcs Characteristic Ofmentioning

confidence: 99%

Autophagy Promotes Porcine Parvovirus Replication and Induces Non-Apoptotic Cell Death in Porcine Placental Trophoblasts

Zhang

Xiong

Zhang

et al. 2019

Viruses

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Autophagy plays important roles in the infection and pathogenesis of many viruses, yet the regulatory roles of autophagy in the process of porcine parvovirus (PPV) infection remain unclear. Herein, we show that PPV infection induces autophagy in porcine placental trophoblasts (PTCs). Induction of autophagy by rapamycin (RAPA) inhibited the occurrence of apoptotic cell death, yet promoted viral replication in PPV-infected cells; inhibition of autophagy by 3-MA or ATG5 knockdown increased cellular apoptosis and reduced PPV replication. Interestingly, we found that in the presence of caspase-inhibitor zVAD-fmk, PPV induces non-apoptotic cell death that was characterized by lysosomal damage and associated with autophagy. Induction of complete autophagy flux by RAPA markedly promoted PPV replication compared with incomplete autophagy induced by RAPA plus bafilomycin (RAPA/BAF) in the early phase of PPV infection (24 h.p.i.). Meanwhile, induction of complete autophagy with RAPA increased lysosomal damage and non-apoptotic cell death in the later phase of PPV infection. Therefore, our data suggest that autophagy can enhance PPV replication and promote the occurrence of lysosomal-damage-associated non-apoptotic cell death in PPV-infected porcine placental trophoblasts.

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The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization

Cited by 30 publications

References 4 publications

N-Acetylcysteine Reverses Antiretroviral-Mediated Microglial Activation by Attenuating Autophagy-Lysosomal Dysfunction

N-Acetylcysteine Reverses Antiretroviral-Mediated Microglial Activation by Attenuating Autophagy-Lysosomal Dysfunction

L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes

Autophagy Promotes Porcine Parvovirus Replication and Induces Non-Apoptotic Cell Death in Porcine Placental Trophoblasts

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