The lipid flippase ATP10B enables cellular lipid uptake under stress conditions.

Pathogenic ATP10B variants have been described in patients with Parkinson's disease and dementia with Lewy body disease, and we previously established ATP10B as a late endo-/lysosomal lipid flippase transporting both phosphatidylcholine (PC) and glucosylceramide (GluCer) from the lysosomal exoplasmic to cytoplasmic membrane leaflet. Since several other lipid flippases regulate cellular lipid uptake, we here examined whether also ATP10B impacts cellular lipid uptake. Transient co-expression of ATP10B with its obligatory subunit CDC50A stimulated the uptake of fluorescently (NBD-) labeled PC in HeLa cells. This uptake is dependent on the transport function of ATP10B, is impaired by disease-associated variants and appears specific for NBD-PC. Uptake of non-ATP10B substrates, such as NBD-sphingomyelin or NBD-phosphatidylethanolamine is not increased. Remarkably, in stable cell lines co-expressing ATP10B/CDC50A we only observed increased NBD-PC uptake following treatment with rotenone, a mitochondrial complex I inhibitor that induces transport-dependent ATP10B phenotypes. Conversely, Im95m and WM-115 cells with endogenous ATP10B expression, present a decreased NBD-PC uptake following ATP10B knockdown, an effect that is exacerbated under rotenone stress. Our data show that the endo-/lysosomal lipid flippase ATP10B contributes to cellular PC uptake under specific cell stress conditions.

Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum.

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy.

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

Contribution of the Presenilins in the cell biology, structure and function of γ-secretase.

γ-Secretase cleavage is essential for many biological processes and its dysregulation is linked to disease, including cancer and Alzheimer's disease. Therefore, understanding the regulation of its activity is of major importance to improve drug design and develop novel therapeutics. γ-Secretase belongs to the family of intramembrane cleaving proteases (i-CLiPs), which cleaves its substrates in a process termed regulated intramembrane proteolysis (RIP). During RIP, type-I transmembrane proteins are first cleaved within their ectodomain by a sheddase and then within their transmembrane domain by γ-secretase. γ-Secretase is composed of four integral membrane proteins that are all essential for its function: presenilin (PSEN), anterior pharynx defective 1 (APH1), nicastrin (NCT) and presenilin enhancer 2 (PEN-2). Given the presence of two PSEN homologues (PSEN1 & 2) and several APH1 isoforms, a heterogeneity exists in cellular γ-secretase complexes. It is becoming clear that each of these complexes has overlapping as well as distinct biological characteristics. This review summarizes our current knowledge on complex formation, trafficking, subcellular localization, interactors and the structure of γ-secretase, with a focus, when possible or known, on the contribution of PSEN1 and PSEN2 herein.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.