Dynamic Measurement of Endosome-Lysosome Fusion in Neurons Using High-Content Imaging.

Endocytosis is a dynamic cellular process that actively transports particles into a cell. Late endosome fusion with the lysosome is a crucial step in the delivery of newly synthesized lysosomal proteins and endocytosed cargo for degradation. Disturbing this step in neurons is associated with neurological disorders. Thus, studying endosome-lysosome fusion in neurons will provide new insight into the mechanisms of these diseases and open new possibilities for therapeutic treatment. However, measuring endosome-lysosome fusion is challenging and time consuming, which limits the research in this area. Here we developed a high throughput method using pH-insensitive dye-conjugated dextrans and the Opera Phenix® High Content Screening System. By using this method, we successfully separated endosomes and lysosomes in neurons, and time-lapse images were collected to capture endosome-lysosome fusion events in hundreds of cells. Both assay set-up and analysis can be completed in an expeditious and efficient manner.

AP2S1 regulates APP degradation through late endosome-lysosome fusion in cells and APP/PS1 mice.

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.

Mystery of methamphetamine-induced autophagosome accumulation in hippocampal neurons: loss of syntaxin 17 in defects of dynein-dynactin driving and autophagosome-late endosome/lysosome fusion.

Methamphetamine (METH), a psychoactive-stimulant facilitates massive accumulation of autophagosomes and causes autophagy-associated neuronal death. However, the underlying mechanisms involving METH-induced auto-phagosome accumulation remain poorly understood. In the current study, autophagic flux was tracked by mRFP-GFP-LC3 adenovirus, 900 µM METH treatment was found to significantly disrupt autophagic flux, which was further validated by remarkable increase of co-localized of LC3 and SQSTM1/p62, enhancement of LC3-II and SQSTM1/p62 protein levels, and massive autophagosome puncta aggregation. With the cycloheximide (CHX) treatment, METH treatment was displayed a significant inhibition of SQSTM1/p62 degradation. Therefore, the mRNAs associated with vesicle degradation were screened, and syntaxin 17 (Stx17) and dynein-dynactin mRNA levels significantly decreased, an effect was proved in protein level as well. Intriguingly, METH induced autophagosome accumulation and autophagic flux disturbance was incredibly retarded by overexpression of Stx17, which was validated by the restoration of the fusion autophagosome-late endosome/lysosome fusion. Moreover, Stx17 overexpression obviously impeded the METH-induced decrease of co-localization of the retrograded motor protein dynein/dynactin and autophagosome-late endosome, though the dynein/dynactin proteins were not involved in autophagosome-late endosome/lysosome fusion. Collectively, our findings unravel the mechanism of METH-induced autophagosome accumulation involving autophagosome-late endosome/lysosome fusion deficiency and that autophagy-enhancing mechanisms such as the overexpression of Stx17 may be therapeutic strategies for the treatment of METH-induced neuronal damage.

Alteration of lysosome fusion and low-grade inflammation mediated by super-low-dose endotoxin.

Subclinical super-low-dose endotoxin LPS is a risk factor for the establishment of low-grade inflammation during the pathogenesis and progression of chronic diseases. However, the underlying mechanisms are not well understood. At the cellular level, a disruption of lysosome fusion with endosomes or autophagosomes may contribute to the potentiation of low-grade inflammation. In this study, we identified that subclinical super-low-dose endotoxin LPS can potently inhibit the process of endosome acidification and lysosome fusion with endosomes or autophagosomes in primary macrophages. Super-low-dose LPS induced the inhibitory phosphorylation of VPS34, thus leading to the disruption of endosome-lysosome fusion. This effect may depend upon the clearance and relocation of Tollip in macrophages by super-low-dose LPS. Consistent with this notion, Tollip-deficient macrophages had constitutively elevated levels of VPS34 inhibitory phosphorylation and constitutive disruption of endosome-lysosome fusion. By employing a skin excision wound-healing model, we observed that Tollip-deficient mice had significantly elevated levels of cell stress and reduced wound repair. This study reveals a novel mechanism responsible for the modulation of endosome-lysosome fusion and low-grade inflammation in innate macrophages.