Objective: Aims of this study include: 1) to generate induced pluripotent stem cell (iPSC)-derived neurons from patients with AP-4-associated HSP; 2) to characterize these neurons with respect to AP-4-related cellular phenotypes; 3) to develop a phenotypic high-throughput screen.

Background: Bi-allelic, loss-of-function variants in subunits of adaptor protein complex 4 (AP-4) lead to four poorly understood forms of childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1) [1]. AP-4 facilitates the selective incorporation of transmembrane cargo proteins into vesicles and mediates their transport [2, 3].

Method: We investigate patient-derived fibroblasts and iPSC-derived neurons using immunocytochemistry, biochemistry and high-content confocal imaging.

Results: To explore the molecular defects underlying AP-4-HSP we investigated 15 patient-derived fibroblast lines and 6 lines of iPSC-derived neurons covering a wide range of AP-4 variants. All patient-derived cells showed reduced levels of AP4E1, a surrogate for levels of AP-4. In all patient-derived fibroblasts the autophagy protein ATG9A, which was recently identified as an AP-4 cargo protein, accumulated in the trans-Golgi network (TGN) and was concomitantly depleted from peripheral sites. Automated quantification of the area of ATG9A signal overlapping with the TGN in relation to the total ATG9A area showed a significant increase in all patient lines. Western blot analysis revealed increased whole cell levels of ATG9A to 3-5x the level of heterozygous or wild-type controls. Looking at the downstream effects of AP-4 loss and ATG9A mislocalization, we find that autophagic flux is present but autophagosome formation in response to starvation is reduced. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47 we find that AP-4 subunit levels are reduced, ATG9A levels are increased and ATG9A accumulates in the TGN. We establish the latter phenotype as a surrogate for AP-4 deficiency in a high-throughput format using automated confocal microscopy.

Conclusion: We identify several cellular phenotypes of AP-4-HSP and establish ATG9A mislocalization as a marker of AP-4 deficiency. Use of iPSC-derived cortical neurons from AP-4-HSP patients in a phenotypic small molecule screen may identify targeted therapies.

References: 1. Ebrahimi-Fakhari, D., et al., AP-4-Associated Hereditary Spastic Paraplegia, in GeneReviews((R)), M.P. Adam, et al., Editors. 2018: Seattle (WA). 2. Hirst, J., C. Irving, and G.H. Borner, Adaptor protein complexes AP-4 and AP-5: new players in endosomal trafficking and progressive spastic paraplegia. Traffic, 2013. 14(2): p. 153-64. 3. Davies, A.K., et al., AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A. Nat Commun, 2018. 9(1): p. 3958.

« Back to 2019 International Congress

MDS Abstracts - https://www.mdsabstracts.org/abstract/adaptor-protein-complex-4-associated-hereditary-spastic-paraplegia-ap-4-hsp-a-paradigm-of-childhood-onset-hereditary-spastic-paraplegia-caused-by-defective-protein-trafficking/