Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Upregulation of APP endocytosis by neuronal aging drives amyloid-dependent synapse loss.

Neuronal aging increases the risk of late-onset Alzheimer's disease. During normal aging, synapses decline, and ß-amyloid (Aß) accumulates intraneuronally. However, little is known about the underlying cell biological mechanisms. We studied neuronal aging using normal-aged brain and aged mouse primary neurons that accumulate lysosomal lipofuscin and show synapse loss. We identified the upregulation of amyloid precursor protein (APP) endocytosis as a neuronal aging mechanism that potentiates APP processing and Aß production in vitro and in vivo. The increased APP endocytosis may contribute to the early endosome enlargement observed in the aged brain. Mechanistically, we showed that clathrin-dependent APP endocytosis requires F-actin and that clathrin and endocytic F-actin increase with neuronal aging. Finally, Aß production inhibition reverts synaptic decline in aged neurons, whereas Aß accumulation, promoted by endocytosis upregulation in younger neurons, recapitulates aging-related synapse decline. Overall, we identify APP endocytosis upregulation as a potential mechanism of neuronal aging and, thus, a novel target to prevent late-onset Alzheimer's disease. This article has an associated First Person interview with the first author of the paper.

A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution.

Alzheimer's disease's established primary trigger is ß-amyloid (Aß) (Mucke and Selkoe, 2012). The amyloid precursor protein (APP) endocytosis is required for Aß generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes depends on its sorting for degradation in lysosomes ( Haass et al., 1992 ; Morel et al., 2013 ; Edgar et al., 2015 ; Ubelmann et al., 2017 ). The following endocytosis assay has been optimized to assess the amyloid precursor protein (APP) endocytosis and degradation by live murine cortical primary neurons ( Ubelmann et al., 2017 ).

Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells.

The established primary trigger of Alzheimer's disease's is ß-amyloid (Aß) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aß generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane ( Koo et al., 1996 ; Ubelmann et al., 2017 ). The following recycling assay has been optimized to assess APP recycling by live murine Neuro2a cells, a neuroblastoma cell line ( Ubelmann et al., 2017 ).