Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease
by Hirst J, Edgar JR, Esteves T, Darios F, Madeo M, Chang J, Roda RH, Dürr A, Anheim M, Gellera C, Li J, Züchner S, Mariotti C, Stevanin G,Blackstone C, Kruer MC, Robinson MS.
Adaptor proteins (AP 1–5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations inAP5Z1, 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.
Ultrastructure of AP5Z1p.Q578* fibroblasts. (A) EM of patient-derived fibroblasts. Note the accumulation of morphologically defined endocytic structures filled with aberrant storage material typified by multiple exaggerated membrane whorls (filled arrow head), belts of striated material (double arrow), fingerprint bodies (arrow) and some intraluminal vesicles (open arrow head). These structures are predominantly surrounded by a single bilayer membrane, and there is clustering of endocytic structures marked by arrowheads with white outline (far left panel; EL, endolysosome). Scale bar = 500 nm. (B) Images were collected from 10 cells and the size of endocytic structures measured (96 for control_1 and 191 for the p.Q578* line); the individual data are shown for endolysosomes (Endolyso), endosomes (Endo) and lysosomes (Lyso) and combined together to show all endocytic structures. Note that no significant difference in the overall size of endocytic structures was revealed. (C) The number of different endocytic structures per unit area of cytoplasm was measured. Images were collected from 10 cells and the number of endocytic structures measured (96 for control_1 and 191 for the p.Q578* line). Note the increase in the number of endolysosomes per unit area of cytoplasm in the p.Q578* line. (D) Examples of AP5Z1p.Q578* fibroblasts showing the clustering of endolysosomes compared with its control. Scale bar = 1 µm.