Lysosomes are central in cell homeostasis and participate in macromolecular degradation, plasma membrane repair, exosome release, cell adhesion/migration, and apoptosis. In cancer, alterations in lysosomal function and spatial distribution may facilitate disease progression. In this study, we show enhanced lysosomal activity in malignant melanoma cells compared with that in normal human melanocytes. Most lysosomes show perinuclear location in melanocytes, while they are more dispersed in melanoma, with retained proteolytic activity and low pH also in the peripheral population. Rab7a expression is lower in melanoma cells than in melanocytes, and by increasing Rab7a, lysosomes are relocated to the perinuclear region in melanoma. Exposure to the lysosome-destabilizing drug L-leucyl-L-leucine methyl ester causes higher damage in the perinuclear subset of lysosomes in melanomas, whereas differences in subpopulation susceptibility cannot be found in melanocytes. Interestingly, melanoma cells recruit the endosomal sorting complex required for transport-III core protein CHMP4B, involved in lysosomal membrane repair, rather than initiate lysophagy. However, when the perinuclear lysosomal position is promoted by Rab7a overexpression or kinesore treatment, lysophagy is increased. In addition, Rab7a overexpression is accompanied by reduced migration capacity. Taken together, the study emphasizes that alterations in lysosomal properties facilitate the malignant phenotype and declares the targeting of lysosomal function as a future therapeutic approach.

Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.

The lysosome is the main unit for degradation and plays important roles in various cellular processes, such as nutrient sensing, cholesterol regulation and cell death. Consequently, altered lysosomal function contributes to, or even causes, several diseases. Lysosomal membrane permeabilization (LMP) and release of lysosomal content to the cytosol can induce cell death, and is implicated in inflammation and neuronal decline in several neurodegenerative diseases. It has also emerged as a potential target in cancer therapy. Due to the detrimental effects of LMP, cells harbor several mechanisms to protect and prevent lysosomal membrane damage. The aim of this thesis was to elucidate how lysosomal membrane stability and repair mechanisms affect cell death and survival.  

We find that lysosomal cholesterol is upregulated in response to an increased load of reactive oxygen species in a Parkinson’s disease cell model, and that augmented cholesterol protects from LMP. However, cholesterol also induces accumulation of α-synuclein and inhibits lysosome-mediated degradation, which can destabilize the lysosomal membrane and accelerate the course of disease. Further, we demonstrate that lysosomal membrane damage is counteracted by a calcium-dependent repair mechanism to prevent LMP. Lysosomes damaged beyond repair are instead sequestered in an autophagosome and degraded by intact lysosomes in a process called lysophagy. As a result, small vesicles containing lysosomal membrane proteins are generated, which we believe are used to restore lysosomal function. We show that malignant cells are more sensitive to LMP, and that they differ in their activation of damage-response mechanisms compared to normal cells. Moreover, in malignant cells, the intracellular position of the lysosomes determines the susceptibility to lysosomal damage. Peripherally located lysosomes are less sensitive, and by relocating lysosomes to the perinuclear area in the cell, we can sensitize lysosomes to LMP induction.  

In summary, this thesis demonstrates the importance of damage-response mechanisms to protect from lysosomal membrane damage and maintain cellular function. It also indicates that targeting of lysosomal stability and repair is a potential therapeutic strategy in both neurodegenerative diseases and in cancer.

Skin pigmentation is controlled by complex crosstalk between melanocytes and keratinocytes and is primarily induced by exposure to ultraviolet (UV) irradiation. Several aspects of UVA-induced signaling remain to be explored. In skin cells, UVA induces plasma membrane damage, which is repaired by lysosomal exocytosis followed by instant shedding of extracellular vesicles (EVs) from the plasma membrane. The released EVs are taken up by neighboring cells. To elucidate the intercellular crosstalk induced by UVA irradiation, EVs were purified from UVA-exposed melanocytes and added to keratinocytes. Transcriptome analysis of the keratinocytes revealed the activation of TGF-beta and IL-6/STAT3 signaling pathways and subsequent upregulation of microRNA (miR)21. EVs induced phosphorylation of ERK and JNK, reduced protein levels of PDCD4 and PTEN, and augment antiapoptotic signaling. Consequently, keratinocyte proliferation and migration were stimulated and UV-induced apoptosis was significantly reduced. Interestingly, melanoma cells and melanoma spheroids also generate increased amounts of EVs with capacity to stimulate proliferation and migration upon UVA. In conclusion, we present a novel intercellular crosstalk mediated by UVA-induced lysosome-derived EVs leading to the activation of proliferation and antiapoptotic signaling via miR21.

O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

Lysosomes are central organelles for cellular degradation and energy homeostasis. In addition, lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal content to the cytosol can initiate programmed cell death. The extent of LMP and available repair mechanisms determine the cell fate after lysosomal damage. In this study, we aimed to investigate the premises for lysosomal membrane repair after LMP and found that lysosomal membrane damage initiated by l-leucyl-l-leucine methyl ester (LLOMe) caused caspase-dependent apoptosis in almost 50% of the cells, while the rest recovered. Immediately after LLOMe addition, lysosomal proteases were detected in the cytosol and the ESCRT-components ALIX and CHMP4B were recruited to the lysosomal membrane. Next, lysophagic clearance of damaged lysosomes was evident and a concentration-dependent translocation of several lysosomal membrane proteins, including LAMP2, to the cytosol was found. LAMP2 was present in small vesicles with the N-terminal protein chain facing the lumen of the vesicle. We conclude that lysophagic clearance of damaged lysosomes results in generation of lysosomal membrane protein complexes, which constitute small membrane enclosed units, possibly for recycling of lysosomal membrane proteins. These lysosomal membrane complexes enable an efficient regeneration of lysosomes to regain cell functionality.

The acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization.

Parkinsons disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated alpha-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), we found that MPP+-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of alpha-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP+-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect alpha-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinsons disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of alpha-synuclein accumulation. (C) 2017 Elsevier GmbH. All rights reserved.

Background: Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-alpha is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-alpha is not yet completely revealed. Methods: Expression of FAP-alpha was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-beta 1. Results: Fibroblast activation protein-a expression was induced by UVR in melanocytes of human skin. The FAP-alpha expression was regulated by UVR-induced release of TGF-beta 1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-alpha mediated ECM degradation and facilitated tumour cell dissemination. Conclusions: Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-beta 1 and FAP-alpha expression, promoting cancer cell dissemination and melanoma metastatic spread.

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca2+-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.

Solar radiation is an important risk factor for skin cancer, the incidence of which is increasing, especially in the fair-skinned populations of the world. While the ultraviolet (UV)B component has direct DNA damaging ability, UVA-induced effects are currently mainly attributed to the production of reactive oxygen species. In our recent study, we compared the effects of UVA and UVB radiation on human keratinocytes and found that UVA-induced plasma membrane damage was rapidly repaired by lysosomal exocytosis, which was detected based on the expression of lysosomal membrane associated protein-1 (LAMP-1) on the plasma membrane of non-permeabilized cells. Later, the keratinocytes died through caspase-8 mediated apoptosis. In contrast, the plasma membranes of keratinocytes exposed to UVB showed no LAMP-1 expression, and, although the cells died by apoptosis, no initial caspase-8 activity was detected. We have also demonstrated the occurrence of UVA-induced lysosomal exocytosis in reconstructed skin and shown the relocation of lysosomes from the center of cells to the vicinity of the plasma membrane. Thus, we suggest that lysosomal exocytosis also occurs in keratinocytes covered by the stratum corneum following exposure to UVA. Our findings provide new insight into the mechanism of UVA-induced skin damage.

Ultraviolet (UV) irradiation is a major environmental carcinogen involved in the development of skin cancer. To elucidate the initial signaling during UV-induced damage in human keratinocytes, we investigated lysosomal exocytosis and apoptosis induction. UVA, but not UVB, induced plasma membrane damage, which was repaired by Ca2+-dependent lysosomal exocytosis. The lysosomal exocytosis resulted in extracellular release of cathepsin D and acid sphingomyelinase (aSMase). Two hours after UVA irradiation, we detected activation of caspase-8, which was reduced by addition of anti-aSMAse. Furthermore, caspase-8 activation and apoptosis was reduced by prevention of endocytosis and by the use of cathepsin inhibitors. We conclude that lysosomal exocytosis is part of the keratinocyte response to UVA and is followed by cathepsin-dependent activation of caspase-8. The findings have implications for the understanding of UV-induced skin damage and emphasize that UVA and UVB initiate apoptosis through different signaling pathways in keratinocytes.

High activity of histone deacetylases (HDACs) has been documented in several types of cancer and may be associated with survival advantage. In a head and neck squamous cell carcinoma cell line, cisplatin-induced apoptosis was augmented by pretreatment with the HDAC inhibitor trichostatin Apoptosis was accompanied by lysosomal membrane permeabilization (LMP), as shown by immunoblotting of the lysosomal marker protease cathepsin B in extracted cytosol and by immunofluorescence. Moreover, LAMP-2 (lysosomal associated membrane protein-2) was translocated from lysosomal membranes and found in a digitonin extractable fraction together with cytosolic proteins and pretreatment with trichostatin A potentiated the release. Overall, protein level of LAMP-2 was decreased during cell death and, interestingly, inhibition of cysteine cathepsins, by the pan-cysteine cathepsin inhibitor zFA-FMK, prevented loss of LAMP-2. The importance of LAMP-2 for lysosomal membrane stability, was confirmed by showing that LAMP-2 knockout MEFs (mouse embryonic fibroblasts) were more sensitive to cisplatin as compared to the corresponding wildtype cells. Trichostatin A reduced lysosomal pH from 4.46 to 4.25 and cell death was prevented when lysosomal pH was increased by NH4Cl, or when inhibiting the activity of lysosomal proteases. We conclude that trichostatin A enhances cisplatin induced cell death by decreasing lysosomal pH, which augments cathepsin activity resulting in reduced LAMP-2 level, and might promote LMP.

Previously, we have shown that TNF-alpha protects iron-exposed J774 macrophages against iron-catalyzed oxidative lysosomal disruption and cell death by increasing reduced glutathione and H-ferritin in cells. Because J774 cells are able to harbor large amounts of iron, which is potentially harmful in a redox-active state, we hypothesized that TNF-alpha-stimulated J774 macrophages will prevent iron-driven oxidative killing of alveolar epithelial A549 cells in co-culture. In the present study, iron trichloride (which is endocytosed by cells as hydrated iron-phosphate complexes) was mainly deposited inside the lysosomes of J774 macrophages, while A549 cells, equally iron exposed, accumulated much less iron. When challenged by oxidants, however, reactive lysosomal iron in A549 cells promoted lysosomal disruption and cell death, particularly in the presence of TNF-alpha. This effect resulted from an elevation in ROS generation by TNF-alpha, while a compensatory upregulation of protective molecules (H-ferritin and/or reduced glutathione) by TNF-alpha was absent. A549 cell death was particularly pronounced when iron and TNF-alpha were present in the conditioned medium during oxidant challenge; thus, iron-driven oxidative reactions in the culture medium were a much greater hazard to A549 cells than those taking place inside their lysosomes. Consequently, the iron chelator, deferoxamine, efficiently prevented A549 cell death when added to the culture medium during an oxidant challenge. In co-cultures of TNF-alpha-stimulated lung cells, J774 macrophages sequestered iron inside their lysosomes and protected A549 cells from oxidative reactions and cell death. Thus, the collective effect of TNF-alpha on co-cultured lung cells was mainly cytoprotective.