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Using Primary Fibroblasts and iPSC-Derived Neurons from Patients with AP-4-associated Hereditary Spastic Paraplegia to Support an Unbiased Autophagy-based Phenotypic Screening for Novel Therapeutic Targets

Applicant Dr. Afshin Saffari
Subject Area Clinical Neurology; Neurosurgery and Neuroradiology
Pediatric and Adolescent Medicine
Term from 2020 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 448402208
 
Final Report Year 2023

Final Report Abstract

The Hereditary Spastic Paraplegias (HSP) are a group of over 80 different neurodegenerative disorders and the most common cause of genetically-determined spastic paralysis. Bi-allelic pathogenic variants in genes encoding subunits of the adaptor protein complex 4 (AP-4) lead to prototypical yet poorly understood forms of complex HSP in children, known as AP-4-associated HSP. This group comprises four distinct entities: SPG47 (AP4B1, OMIM #614066); SPG50 (AP4M1, OMIM #612936); SPG51 (AP4E1, OMIM #613744); and SPG52 (AP4S1, OMIM #614067). Previous work identified the core autophagy protein and lipid scramblase ATG9A as a major cargo of AP-4, linking loss of AP-4 function to defective autophagy. AP-4 deficiency in non-neuronal and neuronal cells leads to an accumulation of ATG9A in the trans-Golgi network (TGN), including in human induced pluripotent stem cell (hiPSC)-derived neurons from AP-4-HSP patients . From this body of work, and overlapping neuronal phenotypes of AP-4 and Atg9a knockout mice, the following working model for AP-4 deficiency emerges : (1) AP-4 is required for trafficking of ATG9A from the TGN; (2) loss-of-function variants in AP-4 subunits lead to a loss of AP-4 function; (3) ATG9A accumulates in the TGN leading to a reduction of axonal delivery of ATG9A; (4) lack of ATG9A at the distal axon impairs autophagy leading to axonal degeneration. Currently, no treatments for AP-4-HSP are available. In this study, we leverage intracellular ATG9A mislocalization as a cellular readout for AP- 4 deficiency to develop a large-scale, automated, multiparametric, unbiased phenotypic small molecule screen for modulators of ATG9A trafficking in patient-derived cellular models. We employed this platform to screen a library of 28,864 novel small molecules in AP-4-deficient patient fibroblasts and identified 503 compounds that re-distribute ATG9A from the TGN to the cytoplasm. Through a series of orthogonal assays in neuronal cells, including differentiated AP4B1KO SH-SY5Y cells and hiPSC-derived neurons from AP4-HSP patients, we defined a series of 5 novel compounds that restore neuronal phenotypes of AP-4-deficiency. In a comprehensive multiparametric analysis, a novel small molecule, termed BCH-HSP-C01, emerged as a lead compound. Target deconvolution strategies using transcriptomic and proteomic profiling revealed that BCH-HSP-C01 modulates intracellular vesicle trafficking and increases autophagic flux, potentially through differential expression of several RAB (Ras-associated binding) proteins. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future studies.

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