Late Lysosomes Research Articles

Microglial LRRK2-mediated NFATc1 attenuates α-synuclein immunotoxicity in association with CX3CR1-induced migration and the lysosome-initiated degradation.

Synucleinopathies refer to a range of neurodegenerative diseases caused by abnormal α-synuclein (α-Syn) deposition, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Their pathogenesis is strongly linked to microglial dysfunction and neuroinflammation, which involves the leucine-rich-repeat kinase 2 (LRRK2)-regulated nuclear factor of activated T-cells (NFAT). Of the NFAT family, NFATc1 has been found to be increasingly translocated into the nucleus in α-syn stimulation. However, the specific role of NFATc1-mediated intracellular signaling in PD remains elusive in regulating microglial functions. In the current study, we crossbred LRRK2 or NFATc1 conditional knockout mice with Lyz2Cre mice to generate mice with microglia-specific deletion of LRRK2 or NFATc1, and by stereotactic injection of fibrillary α-Syn, we generated PD models in these mice. We found that LRRK2 deficiency enhanced microglial phagocytosis in the mice after α-Syn exposure and that genetic inhibition of NFATc1 markedly diminished phagocytosis and α-Syn elimination. We further demonstrated that LRRK2 negatively regulated NFATc1 in α-Syn-treated microglia, in which microglial LRRK2-deficiency facilitated NFATc1 nuclear translocation, CX3CR1 upregulation, and microglia migration. Additionally, NFATc1 translocation upregulated the expression of Rab7 and promoted the formation of late lysosomes, resulting in α-Syn degradation. In contrast, the microglial NFATc1 deficiency impaired CX3CR1 upregulation and the formation of Rab7-mediated late lysosomes. These findings highlight the critical role of NFATc1 in modulating microglial migration and phagocytosis, in which the LRRK2-NFATc1 signaling pathway regulates the expression of microglial CX3CR1 and endocytic degradative Rab7 to attenuate α-synuclein immunotoxicity.

Using a Model Lysosome Membrane to Study Nanomaterial-Membrane Interactions

Some engineered nanomaterials (ENM), phagocytosed by cells and transported to lysosomes can cause the release of hydrolytic enzymes into the cytosol of the cell, resulting in an inflammatory response and cytotoxicity. We designed model lipid systems to study interactions between ENM and lipid membranes to assess ENM-lipid interaction. Liposomes (100 nm) were generated from lipids found in lysosomes: bis(monooleoylglycero)phosphate (BMP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), in a ratio of 2:3:3. Tris-buffered saline (TBS, pH 7.4) and acetate-buffered saline (ABS, pH 4.5) was used to mimic the pH of early and late lysosomes. Liposome structure was evaluated using transmission electron microscopy; all preparations were ∼100 nm in diameter and unilamellar. Liposomes were exposure to two types of titanium dioxide (TiO2) ENM, nanospheres (TNS) and nanobelts (TNB), or zinc oxide (ZnO) nanospheres for 2 hours at 25, 100 or 200 µg/ml. Time-resolved fluorescence anisotropy was performed using the fluorescence probe di-4-ANEPPDHQ and a custom-built time-resolved fluorometer to measure changes in membrane fluidity (reported in cone angle). Liposomes (BMP:DOPC:DOPE) exposed to TNS (100 µg/ml) showed no difference in cone angle compared to control in either buffer. However, TNB exposure produced significant decreases in cone angle in TBS at 200 µg/ml (36.42±0.31° to 32.84±0.33°) and in ABS at both 100 and 200 µg/ml (34.92±0.52° to 34.30±0.83° and 30.88±0.12°). Even though the surface area of TNS was greater than TNB the lack of effect supported that shape was important. ZnO ENM exposed liposomes composed of BMP:DOPC:DOPE showed no significant change in cone angle (100 µg/ml). However, BMP:DOPC:DOPS liposome had a significant decrease in cone angle (37.60±0.35° to 34.12±0.30°), indicating lipid headgroup can influence interactions. Funding: NIH R01ES023209, 1F32ES027324, P20GM103546, P30GM103338 and J Murdock Charitable Trust.

Screening of α-Tocopherol Transfer Protein Sensitive Genes in Human Hepatoma Cells (HepG2).

α-Tocopherol transfer protein (α-TTP) is a ~32 kDa protein expressed mainly in hepatocytes. The major function of the protein is to bind specifically to α-tocopherol and, together, the complex transfers from late lysosomes to the cell membrane. A previous study indicated that some factors might be required in the transferring process. However, there is little information available about the potential transferring factors. In addition, there remains much to learn about other physiological processes which α-TTP might participate in. Thus, in this study a human α-TTP eukaryotic expression vector was successfully constructed and expressed in human hepatoma cells (HepG2). The sensitive genes related to α-TTP were then screened by microarray technology. Results showed that expression of the vector in HepG2 cells led to the identification of 323 genes showing differential expression. The differentially expressed transcripts were divided into four main categories, including (1) cell inflammation; (2) cell cycle and cell apoptosis; (3) cell signaling and gene regulation; and (4) cellular movement. A few cellular movement related transcripts were selected and verified by quantitative real-time PCR. Expressions of some were significantly increased in α-TTP-expressed group, which indicated that these factors were likely to play a role in the transferring process.

Systemic delivery of siRNA by hyaluronan-functionalized calcium phosphate nanoparticles for tumor-targeted therapy.

In this study, hyaluronan (HA)-functionalized calcium phosphate nanoparticles (CaP-AHA/siRNA NPs) were developed for an injectable and targetable delivery of siRNA, which were prepared by coating the alendronate-hyaluronan graft polymer (AHA) around the surface of calcium phosphate-siRNA co-precipitates. The prepared CaP-AHA/siRNA NPs had a uniform spherical core-shell morphology with an approximate size of 170 nm and zeta potential of -12 mV. The coating of hydrophilic HA improved the physical stability of nanoparticles over one month due to the strong interactions between phosphonate and calcium. In vitro experiments demonstrated that the negatively charged CaP-AHA/siRNA NPs could effectively deliver EGFR-targeted siRNA into A549 cells through CD44-mediated endocytosis and significantly down-regulate the level of EGFR expression. Also, the internalized CaP-AHA/siRNA NPs exhibited a pH-responsive release of siRNA, indicating that the acidification of lysosomes probably facilitated the disassembling of nanoparticles and the resultant ions sharply increased the inner osmotic pressure and thus expedited the release of siRNA from late lysosomes into the cytoplasm. Furthermore, in vivo tumor therapy demonstrated that high accumulation of CaP-AHA/siEGFR NPs in tumor led to a significant tumor growth inhibition with a specific EGFR gene silencing effect after intravenous administration in nude mice xenografted with A549 tumor, along with a negligible body weight loss. These results suggested that the CaP-AHA/siRNA NPs could be an effective and safe systemic siRNA delivery system for a RNAi-based tumor targeted therapy strategy.

Antagonistic Control of Lysosomal Fusion by Rab14 and the Lyst‐Related Protein LvsB

While loss of the protein Lyst causes abnormal lysosomes in patients with Chediak-Higashi syndrome, the contribution of Lyst to lysosome biology is not known. Previously we found that the Dictyostelium ortholog of Lyst, LvsB, is a cytosolic protein that associates with lysosomes and post-lysosomes to prevent their inappropriate fusion. Here we provide three lines of evidence that indicate that LvsB contributes to lysosome function by antagonizing the function of DdRab14, a protein that promotes homotypic fusion among lysosomes. (1) Instead of restricting DdRab14 to lysosomes, cells that lack LvsB expand DdRab14 localization to include post-lysosomes. (2) Expression of activated DdRab14 phenocopies the loss of LvsB, causing inappropriate heterotypic fusion between lysosomes and post-lysosomes and their subsequent enlargement. (3) Conversely, expression of inactivated DdRab14 suppresses the phenotype of LvsB null cells and restores their lysosomal size and segregation from post-lysosomes. Our data suggest a scenario where LvsB binds to late lysosomes and promotes the inactivation of DdRab14. This inactivation allows the lysosomes to mature into post-lysosomes for eventual secretion. We propose that human Lyst may function similarly to regulate Rab-dependent fusion of lysosomal compartments.

The involvement of microtubules and actin filaments in the intracellular transport of non-viral gene delivery system

It is known that two cytoskeleton components, microtubules and actins filaments, are required for efficient endocytosis. The relative importance of these two components in the cellular uptake of 2-(dimethylamino)ethyl methacrylate (DMAEMA)-DNA polyplexes was investigated in this study by applying microtubule depolymerising agent, colchicine, and actin polymerising inhibitor, cytochalasin D, in a cell transfection study. The effect of colchicine on transfection efficiency of polyplexes was found to be a time-dependent phenomenon, whereby the level of gene expression was inhibited at early stage, presumably to the disruption of a transport of vesicles along microtubules by colchicine. As time progressed, the level of gene expression was significantly enhanced relative to the control, possibly due to the failure in transport of vesicles from endosomes to late lysosomes, or due to the breakdown of nuclear membrane when mitosis was arrested at metaphase by colchicine. On the other hand, transfection efficiency was significantly reduced at all time points by cytochalasin D, which is considered to primarily affects invagination of vesicles at the early stage of endocytosis by inhibiting actin polymerisation. Further investigation to identify the endocytotic route of DMAEMA polyplexes was conducted applying clathrin- and caveolae- pathways inhibitors in cell transfection study. The results indicate that DMAEMA polyplexes were internalized primarily through clathrin-mediated pathway, with a minor fraction possibly entering cells via a caveolae-mediated pathway.

Trafficking pathway and affectivity on extracellular signal-regulated kinase phosphorylation of anti-tumor fusion protein of epidermal growth factor-adenovirus early region 4 open reading frame 4 protein

To investigate the intracellular trafficking pathway of fusion protein epidermal growth factor-adenovirus early region 4 open reading frame 4 protein (EGF-E4orf4) internalized via epidermal growth factor (EGF) receptor and its affectivity on extracellular signal-regulated kinase (ERK) phosphorylation. MDA-MB-231 and BGC823 cells were incubated with fluorescein isothiocyanate-EGF-E4orf4 or EGF at different time points. The specific molecular mark of early endosome or late lysosome was labeled by indirect immunofluorescence, and then colocalization staining was observed using confocal laser microscopy. The levels of ERK phosphorylation were detected by Western blot. The fluorescent signal of fusion protein EGF-E4orf4 accumulated within the cells and congregated to the perinuclear region. A nucleus localization of the fusion protein was only at MDA-MB-231 cell. Colocalization of EGF-E4orf4 with early endosome/late lysosome was observed. EGF-E4orf4 stimulated ERK phosphorylation, which was most obvious 10 minutes after stimulation, and then gradually attenuated, which was similar to EGF stimulation but with a less decrease. Internalized EGF-E4orf4 can be slowly degraded via endosome-lysosome pathway. The action features of EGF-E4orf4 are remarkably different between MDA-MB-231 and BGC823 cells, which may help explain the differences in its anti-tumor potency and in the special selectivity toward different tumor cells.

Cholesterol-Rich Membrane Rafts and Lyn Are Involved in Phagocytosis during Pseudomonas aeruginosa Infection

The mechanism of phagocytosis of pathogens remains to be fully characterized. We report a novel phagocytosis pathway for Pseudomonas aeruginosa, which is initiated by cholesterol-rich membrane rafts and is dependent on Lyn, primarily an immune regulator with both positive and negative roles. Blocking of Lyn or blocking of cholesterol synthesis significantly inhibited phagocytosis by alveolar macrophages. We found that Lyn, via Src homology 2 and 3 domains, bound to and then activated PI3K and Akt to regulate intracellular routing of the engulfed P. aeruginosa. Further analysis indicates that Lyn and raft components entered in phagosomes and late lysosomes. Finally, respiratory burst was dependent on Lyn and membrane rafts, as confirmed by small interfering RNA and dominant-negative strategies. Our investigations demonstrate that Lyn along with membrane rafts plays a fundamental role in phagocytosis by alveolar macrophages during infection.

The BEACH Protein LvsB Is Localized on Lysosomes and Postlysosomes and Limits Their Fusion with Early Endosomes

The Chediak-Higashi syndrome (CHS) is a genetic disorder caused by the loss of the BEACH protein Lyst. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. Dictyostelium LvsB is the ortholog of mammalian Lyst and is also important for lysosomal function. A knock-in approach was used to tag LvsB with green fluorescent protein (GFP) and express it from its single chromosomal locus. GFP-LvsB was observed on late lysosomes and postlysosomes. Loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. The abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of early endosomal compartments with postlysosomal compartments. The intermixing of compartments resulted in a delayed transit of fluid-phase marker through the endolysosomal system. These results support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early endosomes with postlysosomal compartments.

Pepstatin‐insensitive carboxyl proteinase: A biochemical marker for late lysosomes inAmoeba proteus

In order to find a biochemical marker for late lysosomes, we characterized two cDNAs which were cloned by using a monoclonal antibody (mAb) against lysosomes in Amoeba proteus as a probe. The two cDNAs, a 1.3‐kb cDNA in pBSK‐lys45 and a 1.6‐kb cDNA in pBSK‐lys60, were found to encode proteins homologous to pepstatin‐insensitive carboxyl proteinases (PICPs). E. coli transformed with pBSK‐lys45 produced two immunopositive polypeptides (45 and 43 kDa) and the cDNA in 1274 bases encoded a 44,733‐Da protein (Lys45) of 420 amino acids containing one site for a core oligosaccharide. On the other hand, E. coli transformed with pBSK‐lys60 produced several polypeptides (64, 54, 45, 41, and 37 kDa) reacting with the mAb. The cDNA contained 1629 bases and encoded a 59,231‐Da protein (Lys60) of 530 amino acids containing two sites for asparagine‐linked core oligosaccharides. These two cDNAs showed identities of 60.3% in nucleotide sequences and 23.6% in amino acid sequences. Lys45 and Lys60 appeared to share XXEFQK as...

Involvement of Early and Late Lysosomes in the Degradation of Mannosylated Ligands by Rat Liver Endothelial Cells

The intracellular transport and degradation of endocytosed mannosylated albumin (Man-BSA) was studied in cell cultures of rat liver endothelial cells by subcellular fractionation, fluorescence microscopy, and electron microscopy. The ligand used for subcellular fractionation experiments was labeled with 125 I-labeled tyramine cellobiose or 131I-labeled tyramine cellobiose. The labeled degradation products are trapped in the degradative compartments and may therefore serve as markers for these compartments. Cell fractionation was performed using Nycodenz gradients. The cell fractionation experiments demonstrated that the ligand sequentially occupied three compartments of increasing density. After 15 min it was mainly found in large cisternal organelles that banded in the gradient at about 1.09 g/ml. These organelles were rab5 positive and showed a peripher distribution in the fluorescence microscope. Degradation of ligand started after 30-60 min and this coincided with its transfer to a electron lucent vesicle with a density of 1.12 g/ml. After >1 h, degradation products started to accumulate in perinuclear, electron-dense lysosomes that banded in the gradient at 1.15 g/ml. The density distribution of lysosomal β-acetylglucosaminidase coincided with the densest organelle. The results obtained show that the degradation of ligand takes place sequentially in two types of lysosomes. The early lysosome is an electron-lucent vesicle of low density, whereas the terminal lysosome is an electron-dense organelle with higher density and a more perinuclear distribution. The main degradation of the ligand takes place in the early lysosome. The transfer of ligand and degradation products from the early to the late lysosome is slow. Texas red-labeled ovalbumin (OVA) coincided with lysosomes labeled with OVA-Bodipy 24 h in advance only after 4-6 h.