Lysosomal Rupture Research Articles

Peroxisome proliferator-activated receptor δ rescues xCT-deficient cells from ferroptosis by targeting peroxisomes

Ferroptosis is a recently recognized process of cell death characterized by accumulation of iron-dependent lipid peroxides. Herein, we demonstrate that peroxisome proliferator-activated receptor δ (PPARδ) inhibits ferroptosis of mouse embryonic fibroblasts (MEFs) derived from cysteine/glutamate transporter (xCT)-knockout mice. Activation of PPARδ by the specific ligand GW501516 led to a dose-dependent decrease in ferroptotic cell death triggered by xCT deficiency, along with decreased levels of intracellular iron accumulation and lipid peroxidation. These effects of GW501516 were abolished by PPARδ-targeting small interfering RNA (siRNA) and the PPARδ inhibitor GSK0660, indicating that PPARδ inhibits xCT deficiency-induced ferroptosis. In addition, GW501516-activated PPARδ time- and dose-dependently upregulated catalase expression at both the mRNA and protein levels. This PPARδ-mediated upregulation of catalase was markedly attenuated in cells treated with PPARδ-targeting siRNA and GSK0660, indicating that expression of catalase is dependent on PPARδ. Consistently, the effects of GW501516 on ferroptosis of xCT-deficient MEFs were counteracted in the presence of 3-amino-1,2,4-triazole, a specific inhibitor of catalase, suggesting that catalase is essential for the effect of PPARδ on ferroptosis triggered by xCT deficiency. GW501516-activated PPARδ stabilized peroxisomes through catalase upregulation by targeting peroxisomal hydrogen peroxide-mediated lysosomal rupture, which led to ferroptosis of xCT-deficient MEFs. Collectively, these results demonstrate that PPARδ modulates ferroptotic signals in xCT-deficient MEFs by regulating catalase expression.

Core Hydrophobicity of Supramolecular Nanoparticles Induces NLRP3 Inflammasome Activation.

Designer nanomaterials capable of delivering immunomodulators to specific immune cells have been extensively studied. However, emerging evidence suggests that several of these nanomaterials can nonspecifically activate NLRP3 inflammasomes, an intracellular multiprotein complex controlling various immune cell functions, leading to undesirable effects. To understand what nanoparticle attributes activate inflammasomes, we designed a multiparametric polymer supramolecular nanoparticle system to modulate various surface and core nanoparticle-associated molecular patterns (NAMPs), one at a time. We also investigated several underlying signaling pathways, including lysosomal rupture-cathepsin B maturation and calcium flux-mitochondrial ROS production, to gain mechanistic insights into NAMPs-mediated inflammasome activation. Here, we report that out of the four NAMPs tested, core hydrophobicity strongly activates and positively correlates with the NLRP3 assembly compared to surface charge, core rigidity, and surface hydrophobicity. Moreover, we demonstrate different signaling inclinations and kinetics followed by differential core hydrophobicity patterns with the most hydrophobic ones exhibiting both lysosomal rupture and calcium influx early on. Altogether, this study will help design the next generation of polymeric nanomaterials for specific regulation of inflammasome activation, aiding efficient immunotherapy and vaccine delivery.

Porphyromonas Gingivalis Infection Induces Synaptic Failure via Increased IL-1β Production in Leptomeningeal Cells.

Studies have reported that synaptic failure occurs before the Alzheimer's disease (AD) onset. The systemic Porphyromonas gingivalis (P. gingivalis) infection is involved in memory decline. We previously showed that leptomeningeal cells, covering the brain, activate glial cells by releasing IL-1β in response to systemic inflammation. In the present study, we focused on the impact of leptomeningeal cells on neurons during systemic P. gingivalis infection. The responses of leptomeningeal cells and cortical neurons to systemic P. gingivalis infection were examined in 15-month-old mice. The mechanism of IL-1β production by P. gingivalis infected leptomeningeal cells was examined, and primary cortical neurons were treated with P. gingivalis infected leptomeningeal cells condition medium (Pg LCM). Systemic P. gingivalis infection increased the expression of IL-1β in leptomeninges and reduced the synaptophysin (SYP) expression in leptomeninges proximity cortex in mice. Leptomeningeal cells phagocytosed P. gingivalis resulting in lysosomal rupture and cathepsin B (CatB) leakage. Leaked CatB mediated NLRP3 inflammasome activation inducing IL-1β secretion in leptomeningeal cells. Pg LCM decreased the expression of synaptic molecules, including SYP, which was inhibited by an IL-1 receptor antagonist pre-treatment. These observations demonstrate that P. gingivalis infection is involved in synaptic failure by inducing CatB/NLRP3 inflammasome-mediated IL-1β production in leptomeningeal cells. The periodontal bacteria-induced synaptic damage may accelerate the onset and cognitive decline of AD.

β2-microglobulin triggers NLRP3 inflammasome activation in tumor-associated macrophages to promote multiple myeloma progression

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (β2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m promotes β2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1β (IL-1β) and IL-18. This process depends on activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lack efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Our results provide mechanistic evidence for β2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.

Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity

The maintenance of lysosomal integrity is essential for lysosome function and cell fate. Damaged lysosomes are degraded by lysosomal autophagy, lysophagy. The mechanism underlying lysophagy remains largely unknown; this study aimed to contribute to the understanding of this topic. A cell-based screening system was used to identify novel lysophagy modulators. Triamterene (6-phenylpteridine-2,4,7-triamine) was identified as one of the most potent lysophagy inducers from the screening process. We found that triamterene causes lysosomal rupture without affecting other cellular organelles and increases autophagy flux in HepG2 cells. Damaged lysosomes in triamterene-treated cells were removed by autophagy-mediated pathway, which was inhibited by depletion of the autophagy regulator, ATG5 or SQSTM1. In addition, treatment of triamterene decreased the integrity of lysosome and cell viability, which were rescued by removing the triamterene treatment in HepG2 cells. Hence, our data suggest that triamterene is a novel lysophagy inducer through the disruption of lysosomal integrity.

Biotransformation of soluble-insoluble lanthanum species and its induced NLRP3 inflammasome activation and chronic fibrosis

Soluble lanthanum (La)(Ⅲ) species that have been extensively used as fertilizers in agriculture can potentially get into the human body through foods and environment. Most soluble La(Ⅲ) species can rapidly transform into insoluble La(Ⅲ) species under physiological conditions, however, their potential biological behavior and chronic toxicity are rarely investigated. In the present study, insoluble La(Ⅲ) species formed under physiological condition were identified as nanoscale or microscale particles, and their major components were found to experience biotransformation process upon contact with cells. Insoluble La(Ⅲ) species could adhere to extracellular membrane or be internalized into cells, capable of activating a nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome. The underlying mechanism could be ascribed to K+ efflux and lysosomal rupture because these insoluble La(Ⅲ) species locating at extracellular membrane could reduce the unsaturated fatty acids of cell membrane, leading to potassium (K+) efflux, and those internalized into cells could consume the phospholipids of lysosomal membrane, leading to lysosomal rupture. Mice daily drinking soluble La(Ⅲ) species to mimic drinking tea process for 90 days were found to present NLRP3 inflammasome activation in liver and kidney, as a result of chronic fibrosis, which is potentially correlated to insoluble La(Ⅲ) species formation.

A virulent Bacillus cereus strain from deep-sea cold seep induces pyroptosis in a manner that involves NLRP3 inflammasome, JNK pathway, and lysosomal rupture

ABSTRACT Recent studies indicate that the Bacillus species is distributed in deep-sea environments. However, no specific studies on deep-sea Bacillus cereus have been documented. In the present work, we isolated a B. cereus strain, H2, from the deep-sea cold seep in South China Sea. We characterized the pathogenic potential of H2 and investigated H2-induced death of different types of cells. We found that H2 was capable of tissue dissemination and causing acute mortality in mice and fish following intraperitoneal/intramuscular injection. In vitro studies revealed that H2 infection of macrophages induced pyroptosis and activation of the NLRP3 inflammasome pathway that contributed partly to cell death. H2 infection activated p38, JNK, and ERK, but only JNK proved to participate in H2-triggered cell death. Reactive oxygen species (ROS) and intracellular Ca2+ were essential to H2-induced activation of JNK and NLRP3 inflammasome. In contrast, lysosomal rupture and cathepsins were required for H2-induced NLRP3 inflammasome activation but not for JNK activation. This study revealed for the first time the virulence characteristics of deep-sea B. cereus and provided new insights into the mechanism of B. cereus infection.

A small molecule binding HMGB1 inhibits caspase-11-mediated lethality in sepsis

Caspase-11, a cytosolic lipopolysaccharide (LPS) receptor, mediates lethal immune responses and coagulopathy in sepsis, a leading cause of death worldwide with limited therapeutic options. We previously showed that over-activation of caspase-11 is driven by hepatocyte-released high mobility group box 1 (HMGB1), which delivers extracellular LPS into the cytosol of host cells during sepsis. Using a phenotypic screening strategy with recombinant HMGB1 and peritoneal macrophages, we discovered that FeTPPS, a small molecule selectively inhibits HMGB1-mediated caspase-11 activation. The physical interaction between FeTPPS and HMGB1 disrupts the HMGB1-LPS binding and decreases the capacity of HMGB1 to induce lysosomal rupture, leading to the diminished cytosolic delivery of LPS. Treatment of FeTPPS significantly attenuates HMGB1- and caspase-11-mediated immune responses, organ damage, and lethality in endotoxemia and bacterial sepsis. These findings shed light on the development of HMGB1-targeting therapeutics for lethal immune disorders and might open a new avenue to treat sepsis.

Lysosome-Instructed Self-Assembly of Amino-Acid-Functionalized Perylene Diimide for Multidrug-Resistant Cancer Cells.

Multidrug resistance (MDR) of cancer cells reduces chemotherapeutic efficacy by preventing drug accumulation in the cells through a drug efflux pump and lysosomal sequestration/exocytosis. Herein, to overcome such anticancer resistance, lysosome-targeted self-assembly of perylene diimide (PDI) derivatives is presented as a powerful strategy for effective and selective anticancer therapy. Stimulated by the lysosomal low pH, the amphiphilic PDI derivatives functionalized with amino acids (PDI-AAs) construct fibrous self-assembled structures inside the lysosomes, causing cancer cell apoptosis by lysosomal rupture. In contrast, negligible apoptosis was observed from normal cells by PDI-AA. The agglomerated fibrous assemblies were not removed by lysosomal exocytosis, thereby displaying a 10.7-fold higher anticancer efficacy on MDR cancer cells compared to a doxorubicin chemotherapeutic agent. The MDR-circumventing capability, along with high selectivity toward cancer cells, supports PDI-AAs as potential candidates for the treatment of MDR cancer cells by lysosome-targeted self-assembly.

Photo-responsive prodrug nanoparticles for efficient cytoplasmic delivery and synergistic photodynamic-chemotherapy of metastatic triple-negative breast cancer

Triple-negative breast cancer (TNBC) have been considered as the most malignant subtype of breast cancer with leading incidence and mortality among females. Herein, photo-responsive prodrug nanoparticles (AlP/CPT-NPs) were designed with efficient cytoplasmic delivery of anti-cancer agent for cooperative photodynamic-chemotherapy. AlP/CPT-NPs were prepared using photosensitizer Al(III) phthalocyanine chloride disulfonic acid (AlP) and ROS-activatable camptothecin prodrug (CPT-PD). AlP/CPT-NPs could induce intracellular 1O2 generation upon light exposure, which not only initiate immediate disassembly of AlP/CPT-NPs but also promote cytoplasmic delivery of CPT through 1O2-mediated lysosomal rupture. The released intracellular CPT could be translocated into nuclei in only 5 min post-irradiation. Consequently, AlP/CPT-NPs efficiently suppressed the tumor growth and metastasis of TNBC in a spatiotemporally controlled manner, providing a promising option for effective treatment of metastatic TNBC. Statement of significanceBreast cancer is a complex disease with leading incidence among females, in which triple-negative breast cancer (TNBC) is considered as the most malignant subtype with increased risk of resistance, recurrence and metastasis. Herein, we designed photo-responsive prodrug nanoparticles (AlP/CPT-NPs) for synergistic treatment of metastatic TNBC. Upon 660 nm light exposure, the 1O2 generated by AlP/CPT-NPs could initiate immediate disassembly of AlP/CPT-NPs and further promote cytoplasmic delivery of the therapeutic payloads (camptothecin, CPT). The prepared AlP/CPT-NPs induced potent in vivo phototherapeutic damage through photodynamic-chemotherapy, resulting in complete tumor ablation with metastasis suppression.

Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

Gout is a common inflammatory arthritis caused by the deposition of monosodium urate (MSU) crystals in the joints. This activates the macrophages into a proinflammatory state by inducing NLRP3-dependent interleukin-1β (IL-1β) secretion, resulting in neutrophil recruitment. Soluble decoy receptor 3 (DcR3) is an immune modulator and can exert biological functions via decoy and non-decoy actions. Previously, we showed that DcR3 suppresses lipopolysaccharides (LPS)- and virus-induced inflammatory responses in the macrophages and promotes the macrophages into the M2 phenotype. In this study, we clarified the actions of DcR3 and its non-decoy action motif heparin sulfate proteoglycan (HSPG) binding domain (HBD) in the MSU crystal-induced NLRP3 inflammasome activation in the macrophages and in mice. In bone marrow-derived macrophages, THP-1 and U937 cells, we found that the MSU crystal-induced secretion of IL-1β and activation of NLRP3 were suppressed by both DcR3.Fc and HBD.Fc. The suppression of the MSU-induced NLRP3 inflammasome activation is accompanied by the inhibition of lysosomal rupture, mitochondrial production of the reactive oxygen species (ROS), expression of cathepsins, and activity of cathepsin B, without affecting the crystal uptake and the expression of NLRP3 or pro-IL-1β. In the air pouch mice model of gout, MSU induced less amounts of IL-1β and chemokines secretion, an increased M2/M1 macrophage ratio, and a reduction of neutrophil recruitment in DcR3-transgenic mice, which expresses DcR3 in myeloid cells. Similarly, the mice intravenously treated with DcR3.Fc or HBD.Fc displayed less inflammation response. These findings indicate that HBD of DcR3 can reduce MSU crystal-induced NLRP3 inflammasome activation via modulation of mitochondrial and lysosomal functions. Therefore, we, for the first time, demonstrate a new therapeutic potential of DcR3 for the treatment of gout.

A Virus-Spike Tumor-Activatable Pyroptotic Agent.

Invoking the occurrence of pyroptosis is an emerging strategy for the treatment of cancer. However, the practical applications of pyroptosis for cancer therapy are currently hindered due to the lack of tumor-specific and efficient pyroptotic agents in vivo. Herein, a virus-spike tumor-activatable pyroptotic agent (VTPA) for cancer-specific therapy is reported. The VTPA is composed of an organosilica coated iron oxide nanoparticle core and spiky manganese dioxide protrusions, which can readily accumulate in tumor after systemic administration, facilitate the tumor intracellular lysosomal rupture, and be degraded by tumor over-expressed intracellular glutathione (GSH) to release Mn ions and iron oxide nanoparticles (IONPs) for the synergetic activation of nucleotide binding oligomerization domain-like receptors protein 3 (NLRP3) inflammasomes. Consequently, the activation of NLRP3 inflammasomes and the release of lactate dehydrogenase of tumor cells are observed after the treatment of VTPA, resulting in a specific pyroptotic cell death. To our best knowledge, the structure-dependent and tumor intracellular GSH activatable pyroptotic agents represent the first demonstration of cancer-specific pyroptosis in vivo, providing a novel paradigm for the development of next-generation cancer-specific pyroptotic nanomedicine.

Activation mechanisms of inflammasomes by bacterial toxins.

Inflammasomes are cytosolic innate immune complexes, which assemble in mammalian cells in response to microbial components and endogenous danger signals. A major family of inflammasome activators is bacterial toxins. Inflammasome sensor proteins, such as the nucleotide-binding oligomerisation domain-like receptor (NLR) family members NLRP1b and NLRP3, and the tripartite motif family member Pyrin+ efflux triggered by pore-forming toxins or by other toxin-induced homeostasis-altering events such as lysosomal rupture. Pyrin senses perturbation of host cell functions induced by certain enzymatic toxins resulting in impairment of RhoA GTPase activity. Assembly of the inflammasome complex activates the cysteine protease caspase-1, leading to the proteolytic cleavage of the proinflammatory cytokines IL-1β and IL-18, and the pore-forming protein gasdermin D causing pyroptosis. In this review, we discuss the latest progress in our understanding on the activation mechanisms of inflammasome complexes by bacterial toxins and effector proteins and explore avenues for future research into the relationships between inflammasomes and bacterial toxins.

Macrophage Autophagy and Silicosis: Current Perspective and Latest Insights.

Silicosis is an urgent public health problem in many countries. Alveolar macrophage (AM) plays an important role in silicosis progression. Autophagy is a balanced mechanism for regulating the cycle of synthesis and degradation of cellular components. Our previous study has shown that silica engulfment results in lysosomal rupture, which may lead to the accumulation of autophagosomes in AMs of human silicosis. The excessive accumulation of autophagosomes may lead to apoptosis in AMs. Herein, we addressed some assumptions concerning the complex function of autophagy-related proteins on the silicosis pathogenesis. We also recapped the molecular mechanism of several critical proteins targeting macrophage autophagy in the process of silicosis fibrosis. Furthermore, we summarized several exogenous chemicals that may cause an aggravation or alleviation for silica-induced pulmonary fibrosis by regulating AM autophagy. For example, lipopolysaccharides or nicotine may have a detrimental effect combined together with silica dust via exacerbating the blockade of AM autophagic degradation. Simultaneously, some natural product ingredients such as atractylenolide III, dioscin, or trehalose may be the potential AM autophagy regulators, protecting against silicosis fibrosis. In conclusion, the deeper molecular mechanism of these autophagy targets should be explored in order to provide feasible clues for silicosis therapy in the clinical setting.

β <sub>2</sub>-Microglobulin - A Trigger for NLRP3 Inflammasome Activation in Tumor-Associated Macrophages Promoting Multiple Myeloma Progression

As significant constituents of the tumor microenvironment in multiple Myeloma (MM), pro-inflammatory macrophages are key promoters of disease progression, bone destruction, and immune-impairment. Consequently, the identification of endogenous mediators of these inflammatory processes can open novel therapeutic avenues against major pathological features of MM. Here, we identify beta-2-microglobulin (β2m) as an important driver in the initiation of inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m in patient-derived MAMs promoted β2m amyloid aggregation, resulting in lysosomal rupture and ultimately in the production of active interleukin (IL)-1β and IL-18. Interestingly, this process strictly depended on the activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lacked efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogated inflammasome activation in a murine MM model. Finally, we demonstrate that specific disruption of NLRP3 or IL-18 diminished tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Taken together our results provide novel mechanistic evidence for β2m’s role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 is a potential novel therapeutic approach to combat this severe malignancy.

Inhibition of Different Pain Pathways Attenuates Oxidative Stress in Glial Cells: A Mechanistic View on Neuroprotective Effects of Different Types of Analgesics.

Neuropathic pain results from trauma or diseases affecting the central nervous system (CNS) and triggers a cascade of events in different CNS parts that eventually lead to oxidative injury. This study was aimed to investigate the protective effects of some selected analgesics in neuropathic pain-induced oxidative damage in the isolated glial cells of the rat brain. In this experiment, rats were randomly divided into 5 main groups. Rats in group 1 received no medication, whereas rats in groups 2 to 5 received ASA (aspirin), celecoxib, morphine, and etanercept daily, respectively. Each main group divides into 3 subgroups: normal, sham, and neuropathic pain model rats. The glial cells of the rat brain were isolated at different time points. Our results demonstrate that neuropathic pain induces ROS generation as the major cause of mitochondrial membrane potential collapse (%∆Ψm) and lysosomal membrane rupture, which result in oxidative damage of the glial cells. In addition, ASA and celecoxib had protective effects on the neuropathic pain-induced oxidative stress markers, including ROS production, mitochondrial membrane potential collapse, and lysosomal membrane leakiness at different time points. Furthermore, the oxidative damage markers were significantly decreased by morphine and etanercept in all investigated days. Since arachidonic acid metabolites and TNF-α are produced during neuropathic pain and inflammation, it can be concluded that the inhibition of the substances production or inhibition of the ligands binding with their receptors would help to decrease the destructive effects of neuropathic pain in the glial cells of rat brain.

TP53-induced glycolysis and apoptosis regulator (TIGAR) ameliorates lysosomal damage in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-mediated mouse model of Parkinson’s disease

The progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) correlates with rupture of lysosome in Parkinson’s disease (PD). It has been found that TP53-induced glycolysis and apoptosis regulator (TIGAR) has been attributed to the regulation of metabolic pathways and neuroprotective effect. In the present study, we showed in a mouse model that 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) caused lysosomal damage and DA neurons loss in the SNpc. MPTP only induced SP1-mediated TIGAR upregulation in the early stage of neurotoxin-induced pathology, and this compensatory mechanism was not enough to maintain normal lysosomal function. MPTP significantly decreased the levels of NADPH and GSH, and the effects were ameliorated by the expression of exogenous TIGAR but execerbated by knockdown of TIAGR. TIGAR or NADPH alleviated oxidative stress, rescued lysosomal dysfunction and attenuated DA neurons degeneration. Overexpression of TIGAR or NADPH supplement inhibited MPP+-mediated reactive oxygen species (ROS), lysosomal membrane permeabilization (LMP) and autophagic flux impairment in PC12 cells. Together, these findings suggest that TIGAR reduces MPTP-mediated oxidative stress, lysosomal depletion and DA neuron damage.

ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6 These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Calciprotein particle-induced cytotoxicity via lysosomal dysfunction and altered cholesterol distribution in renal epithelial HK-2 cells

Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

Apoptotic Neuron-Derived Histone Amyloid Fibrils Induce α-Synuclein Aggregation.

Cell-to-cell transfer of α-synuclein (αS) is increasingly thought to play an important role in propagation of αS pathology, but mechanisms responsible for formation of initial αS seeds and factors facilitating their propagation remain unclear. We previously demonstrated that αS aggregates are formed rapidly in apoptotic neurons and that interaction between cytoplasmic αS and proaggregant nuclear factors generates seed-competent αS. We also provided initial evidence that histones have proaggregant properties. Since histones are released from cells undergoing apoptosis or cell stress, we hypothesized that internalization of histones into αS expressing cells could lead to intracellular αS aggregation. Here using mCherry-tagged histone, we show that nuclear extracts from apoptotic cells can induce intracellular αS inclusions after uptake into susceptible cells, while extracts from non-apoptotic cells did not. We also demonstrate that nuclear extracts from apoptotic cells contained histone-immunoreactive amyloid fibrils. Moreover, recombinant histone-derived amyloid fibrils are able to induce αS aggregation in cellular and animal models. Induction of αS aggregation by histone amyloid fibrils is associated with endocytosis-mediated rupture of lysosomes, and this effect can be enhanced in cells with chemically induced lysosomal membrane defects. These studies provide initial descriptions of the contribution of histone amyloid fibrils to αS aggregation.