James R. Edgar

Hrs‐ and CD63‐Dependent Competing Mechanisms Make Different Sized Endosomal Intraluminal Vesicles

Multivesicular endosomes/bodies (MVBs) contain intraluminal vesicles (ILVs) that bud away from the cytoplasm. Multiple mechanisms of ILV formation have been identified, but the relationship between different populations of ILVs and MVBs remains unclear. Here, we show in HeLa cells that different ILV subpopulations can be distinguished by size. EGF stimulation promotes the formation of large ESCRT‐dependent ILVs, whereas depletion of the ESCRT‐0 component, Hrs, promotes the formation of a uniformly sized population of small ILVs, the formation of which requires CD63. CD63 has previously been implicated in ESCRT‐independent sorting of PMEL in MVBs and transfected PMEL is present on the small ILVs that form on Hrs depletion. Upregulation of CD63‐dependent ILV formation by Hrs depletion indicates that Hrs and CD63 regulate competing machineries required for the generation of distinct ILV subpopulations. Taken together our results indicate that ILV size is influenced by their cargo and mechanism of formation and suggest a competitive relationship between ESCRT‐dependent and ‐independent mechanisms of ILV formation within single MVBs.

Interaction between AP-5 and the hereditary spastic paraplegia proteins SPG11 and SPG15

The adaptor protein complex AP-5 is stably associated with the hereditary spastic paraplegia proteins SPG11 and SPG15 in an apparently equimolar ratio to form a new type of coat. Results suggest that SPG15 facilitates the docking of the coat onto late endosomal/lysosomal membranes and that SPG11 (possibly together with SPG15) acts as a scaffold.

Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease

Adaptor proteins (AP 1–5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and ‘fingerprint bodies’. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs.

ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation

ABSTRACT Intracellular amyloid-β (Aβ) accumulation is a key feature of early Alzheimers disease and precedes the appearance of Aβ in extracellular plaques. Aβ is generated through proteolytic processing of amyloid precursor protein (APP), but the intracellular site of Aβ production is unclear. APP has been localized to multivesicular bodies (MVBs) where sorting of APP onto intraluminal vesicles (ILVs) could promote amyloidogenic processing, or reduce Aβ production or accumulation by sorting APP and processing products to lysosomes for degradation. Here, we show that APP localizes to the ILVs of a subset of MVBs that also traffic EGF receptor (EGFR), and that it is delivered to lysosomes for degradation. Depletion of the endosomal sorting complexes required for transport (ESCRT) components, Hrs (also known as Hgs) or Tsg101, inhibited targeting of APP to ILVs and the subsequent delivery to lysosomes, and led to increased intracellular Aβ accumulation. This was accompanied by dramatically decreased Aβ secretion. Thus, the early ESCRT machinery has a dual role in limiting intracellular Aβ accumulation through targeting of APP and processing products to the lysosome for degradation, and promoting Aβ secretion. Highlighted Article: The early ESCRT machinery has a dual role in limiting intracellular Aβ accumulation through targeting of APP and its processing products to lysosomes for degradation, and promoting Aβ secretion.

Tetherin is an exosomal tether

Exosomes are extracellular vesicles that are released when endosomes fuse with the plasma membrane. They have been implicated in various functions in both health and disease, including intercellular communication, antigen presentation, prion transmission, and tumour cell metastasis. Here we show that inactivating the vacuolar ATPase in HeLa cells causes a dramatic increase in the production of exosomes, which display endocytosed tracers, cholesterol, and CD63. The exosomes remain clustered on the cell surface, similar to retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, we knocked it out and found a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. This phenotype could be rescued by wild-type tetherin but not tetherin lacking its GPI anchor. We propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions. DOI: http://dx.doi.org/10.7554/eLife.17180.001

Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.

Fractionation profiling is a universal method for the rapid proteomic characterization of membrane vesicles and protein particles. Although initially developed for the analysis of clathrin-coated vesicles, it also provides a versatile tool for generating high-resolution protein-interaction maps, allowing detailed analysis of protein complexes.

Defects in ER–endosome contacts impact lysosome function in hereditary spastic paraplegia

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER–endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell–derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER–endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking

EpsinR and gadkin are two components of intracellular clathrin-coated vesicles whose precise functions are unclear. Rapid depletion of each protein from the available pool using the knocksideways method strongly inhibited the production of intracellular clathrin-coated vesicles, providing new insights into the functions of both proteins.

Complicated spastic paraplegia in patients with AP5Z1 mutations (SPG48)

Objective: Biallelic mutations in the AP5Z1 gene encoding the AP-5 ζ subunit have been described in a small number of patients with hereditary spastic paraplegia (HSP) (SPG48); we sought to define genotype–phenotype correlations in patients with homozygous or compound heterozygous sequence variants predicted to be deleterious. Methods: We performed clinical, radiologic, and pathologic studies in 6 patients with biallelic mutations in AP5Z1. Results: In 4 of the 6 patients, there was complete loss of AP-5 ζ protein. Clinical features encompassed not only prominent spastic paraparesis but also sensory and motor neuropathy, ataxia, dystonia, myoclonus, and parkinsonism. Skin fibroblasts from affected patients tested positive for periodic acid Schiff and autofluorescent storage material, while electron microscopic analysis demonstrated lamellar storage material consistent with abnormal storage of lysosomal material. Conclusions: Our findings expand the spectrum of AP5Z1-associated neurodegenerative disorders and point to clinical and pathophysiologic overlap between autosomal recessive forms of HSP and lysosomal storage disorders.

Molecular Mechanisms of Disease Pathogenesis Differ in Krabbe Disease Variants

Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full‐length human GALC and used these to monitor the trafficking and processing of GALC variants in cell‐based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post‐translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies.