Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.

Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of ␤-amyloid (A␤) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The A␤ peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases. The endocytic system has been implicated in the cleavages leading to the formation of A␤. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated A␤ is released into the extracellular milieu are not clear. Here, we show that ␤-cleavage occurs in early endosomes followed by routing of A␤ to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of A␤ peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD. multivesicular bodies ͉ rafts ͉ amyloid precursor protein ͉ ␤-secretase ͉ endocytosis A lzheimer's disease (AD) is a late-onset neurological disorder with progressive loss of memory and cognitive abilities as a result of excessive neurodegeneration (1). AD is characterized by extracellular aggregates of ␤-amyloid (A␤) peptides known as amyloid plaques (2). The A␤ peptide is derived from the sequential processing of the amyloid precursor protein (APP) by ␤-and ␥-secretases. ␤-secretase [(␤-APP cleaving enzyme (BACE)] is a type-1 transmembrane aspartyl protease and is mainly localized to endosomes, lysosomes and the transGolgi network (3). ␥-Secretase is a multicomponent complex that is composed of presenilin-1͞presenilin-2, nicastrin, Aph-1, and PEN-2 (4) and is localized to the early secretory (5, 6) and the endocytic compartments (7,8). Nonamyloidogenic processing of APP involves ␣-secretase that cleaves APP inside the A␤ region, giving rise to the ␣-cleaved ectodomain, thus precluding the formation of A␤ (9). Hence, the availability of APP to either ␣-or ␤-secretase determines whether A␤ peptide will be generated. Lateral organization of membranes (10) and subcellular localization (11, 12) of the substrate and the secretases have been documented to regulate A␤ generation. Recent work suggests that ␤-secretase associates with lipid rafts, liquid-ordered domains in the membrane (13,14), and that integrity of raft domains is required for ␤-cleavage of APP to occur (ref. 10; see, however, ref. 15). ␣-Cleavage, in contrast, occurs outside raft domains (10). The ␥-secretase complex is also raft-associated (16); hence, amyloidogenic processing of APP could occur in clustered raft domains to generate A␤ (10). Inhibition of endocytosis reduces ␤-cleavage but not ␣-cleavage, suggesting that ␤-cleavage mainly occurs in endosomes (10,11,(17)(18)(19). Accumulation of A␤ peptides in extracellular...

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Many drug targets are localized to particular subcellular compartments, yet current drug design strategies are focused on bioavailability and tissue targeting and rarely address drug delivery to specific intracellular compartments. Insights into how the cell traffics its constituents to these different cellular locations could improve drug design. In this Review, we explore the fundamentals of membrane trafficking and subcellular organization, as well as strategies used by pathogens to appropriate these mechanisms and the implications for drug design and delivery.

No abstract

The triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial innate immune receptor associated with a lethal form of early, progressive dementia, Nasu-Hakola disease, and with an increased risk of Alzheimer's disease. Microglial defects in phagocytosis of toxic aggregates or apoptotic membranes were proposed to be at the origin of the pathological processes in the presence of Trem2 inactivating mutations. Here, we show that TREM2 is essential for microglia-mediated synaptic refinement during the early stages of brain development. The absence of Trem2 resulted in impaired synapse elimination, accompanied by enhanced excitatory neurotransmission and reduced long-range functional connectivity. Trem2 mice displayed repetitive behavior and altered sociability. TREM2 protein levels were also negatively correlated with the severity of symptoms in humans affected by autism. These data unveil the role of TREM2 in neuronal circuit sculpting and provide the evidence for the receptor's involvement in neurodevelopmental diseases.

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165-3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective.generalized polarization value ͉ giant unilamellar vesicle ͉ membrane organization ͉ lipid sorting ͉ lipid raft T he lipid raft hypothesis postulates that selective interactions among sphingolipids, cholesterol, and membrane proteins contribute to lateral membrane heterogeneity (1). A tenet of the model is that small, dynamic cholesterol-sphingolipid-enriched assemblies can be induced to coalesce into larger, more stable structures through clustering of domain components (2). Although experimental data support cholesterol-dependent nano-scale membrane heterogeneity (3-8) and selective domain formation upon raft cross-linking (9-12), the mechanisms that govern such associations in cell membranes remain unclear.On the molecular level, a key feature that is thought to contribute to raft assembly is the propensity of cholesterol to pack tightly with saturated acyl chains of lipids causing them to adopt an extended conformation (13,14). In multi-component model membranes (n Ͼ 2), this interaction can lead to microscopically separate fluid membrane phases: the liquid-ordered (Lo) phase, enriched in saturated (sphingo-)lipids and cholesterol in a highly condensed state, and the liquid-disordered (Ld) phase, enriched in unsaturated glycerophospholipid in a disordered state (15)(16)(17).Several features of the Lo phase in model membranes correspond to the predicted properties of lipid rafts in cel...

Recent reports indicate that a growing number of intracellular proteins are not only prone to pathological aggregation but can also be released and "infect" neighboring cells. Therefore, many complex diseases may obey a simple model of propagation where the penetration of seeds into hosts determines spatial spread and disease progression. We term these proteins prionoids, as they appear to infect their neighbors just like prions--but how can bulky protein aggregates be released from cells and how do they access other cells? The widespread existence of such prionoids raises unexpected issues that question our understanding of basic cell biology.