Intracellular Ion Changes Induced by the Exposure to Beta-Amyloid Can Be Explained by the Formation of Channels in the Lysosomal Membranes

In this manuscript, we reassess the data on beta-amyloid-induced changes of intracellular ions concentrations published previously by Abramov et al. (2003, 2004). Their observations made using high-resolution confocal microscopy with fast temporal resolution of images formed by fluorescent ion-sensitive fluorescent probes in living cells represent an unequivocal support for the amyloid channel theory. However, closer look reveals multiple facts which cannot be explained by channel formation in plasma membrane. Recently proposed amyloid degradation toxicity hypothesis provides the interpretation to these facts by considering that channels are formed in the lysosomal membranes.

Membrane targeting activates Leucine-rich repeat kinase 2 with differential effects on downstream Rab activation

ABSTRACTGenetic variation at the Leucine-rich repeat kinase 2 (LRRK2) locus contributes to risk of familial and sporadic Parkinson’s disease. Recent data have shown a robust association between localization to various membranes of the endolysosomal system and LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes are still poorly understood. Here we artificially direct LRRK2 to six different membranes within the endolysosomal system. We demonstrate that LRRK2 is activated and able to phosphorylate three of its Rab substrates (Rab10, Rab12 and Rab29) at each compartment. However, we report differing localization of pRab10 and pRab12 at the lysosomal and Golgi membranes. Specifically, we found that pRab10 colocalizes with a sub-population of perinuclear LRRK2-positive Golgi/lysosomal compartments whereas pRab12 localized to all LRRK2-positive Golgi/lysosomal membranes across the cell. When organelle positioning is manipulated by sequestering lysosomes to the perinuclear area, pRab10 colocalization with LRRK2 significantly increases. We also show recruitment of JIP4, a pRab10 effector that we have recently linked to LYTL, after trapping LRRK2 at various membranes. Taken together, we demonstrate that the association of LRRK2 to membranous compartments is sufficient for its activation and Rab phosphorylation independent of membrane identity. Our system also identifies a potential mechanism underlying the distinct relationships between LRRK2 and its substrates Rab10 and Rab12.

The Lysosomotropic Activity of Hydrophobic Weak Base Drugs is Mediated via Their Intercalation into the Lysosomal Membrane

Lipophilic weak base therapeutic agents, termed lysosomotropic drugs (LDs), undergo marked sequestration and concentration within lysosomes, hence altering lysosomal functions. This lysosomal drug entrapment has been described as luminal drug compartmentalization. Consistent with our recent finding that LDs inflict a pH-dependent membrane fluidization, we herein demonstrate that LDs undergo intercalation and concentration within lysosomal membranes. The latter was revealed experimentally and computationally by (a) confocal microscopy of fluorescent compounds and drugs within lysosomal membranes, and (b) molecular dynamics modeling of the pH-dependent membrane insertion and accumulation of an assortment of LDs, including anticancer drugs. Based on the multiple functions of the lysosome as a central nutrient sensory hub and a degradation center, we discuss the molecular mechanisms underlying the alteration of morphology and impairment of lysosomal functions as consequences of LDs’ intercalation into lysosomes. Our findings bear important implications for drug design, drug induced lysosomal damage, diseases and pertaining therapeutics.

In Vitro Cytotoxicity Evaluation of the Magnéli Phase Titanium Suboxides (TixO2x−1) on A549 Human Lung Cells

The use of titanium suboxides, known as Magnéli phase TiOx, is expected to increase in the near future due to their desirable properties. In order to use Magnéli phase TiOx nanoparticles safely, it is necessary to know how nanoparticles interact with biological systems. In this study, the cytotoxicity of three different Magnéli TiOx nanoparticles was evaluated using human lung A549 cells and the results were compared with hazard data on two different TiO2 nanoparticles whose biological interactions have already been extensively studied. After A549 cells were exposed to nanoparticles, the metabolic activity was measured by the Resazurin assay, the amount of cellular proteins was measured by the Coomassie Blue assay, and lysosomal integrity was measured by the Neutral Red Uptake assay. In order to investigate possible modes of particle actions, intracellular Ca2+ level, reactive oxygen species (ROS) production, and photo-oxidative disruptions of lysosomal membranes were assessed. All experiments were performed in serum-containing and in serum-deprived cell culture mediums. In addition, the photocatalytic activity of Magnéli TiOx and TiO2 nanoparticles was measured. The results show that Magnéli TiOx nanoparticles increase intracellular Ca2+ but not ROS levels. In contrast, TiO2 nanoparticles increase ROS levels, resulting in a higher cytotoxicity. Although Magnéli TiOx nanoparticles showed a lower UV-A photocatalytic activity, the photo-stability of the lysosomal membranes was decreased by a greater extent, possibly due to particle accumulation inside lysosomes. We provide evidence that Magnéli TiOx nanoparticles have lower overall biological activity when compared with the two TiO2 formulations. However, some unique cellular interactions were detected and should be further studied in line with possible Magnéli TiOx application. We conclude that Magnéli phase nanoparticles could be considered as low toxic material same as other forms of titanium oxide particles.

LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson’s disease and Crohn’s disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.