High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia

Abstract

Adaptor protein complex 4 (AP-4)-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1, which constitute the four subunits of this obligate complex. While the diagnosis of AP-4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by AP-4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z’-factor robust > 0.3, strictly standardized mean difference > 3). The ‘ATG9A ratio’ is increased in fibroblasts of 18 well-characterized AP-4-associated hereditary spastic paraplegia patients (mean: 1.54 ± 0.13 vs. 1.21 ± 0.05 (standard deviation) in controls) and receiver-operating-characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849–0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect AP-4 function. Our findings establish the ‘ATG9A ratio’ as a diagnostic marker of AP-4-associated hereditary spastic paraplegia.

Ap4b1-knockout mouse model of hereditary spastic paraplegia type 47 displays motor dysfunction, aberrant brain morphology and ATG9A mislocalization

“…In immunohistochemical investigation of numerous CNS locations from Ap4b1 (−/−) mice described here, the retention of ATG9A at the TGN is a distinct pathological hallmark, and is a good diagnostic predictor of AP-4 deficient disorders in patient cells. 52 As trafficking of ATG9A to distal compartments of neurons is disrupted in AP-4 deficiency syndromes, neurons with longer axons are more likely to be affected by ATG9A deprivation and the resulting reduction in autophagosome biogenesis and distal axon formation. 53-55 As murine motor neuron axons are shorter than human axons, ATG9A may better reach distal neuronal compartments in the mouse nervous system; for example, via non-AP-4 mediated means—either by incorporation into other AP complexes such as AP-1 and AP-2, 56 or by passive diffusion.…”

Intrathecal AAV9/AP4M1 gene therapy for hereditary spastic paraplegia 50 shows safety and efficacy in preclinical studies

“…Ebrahimi-Fakhari's group tested the AAV2/AP4M1 vector in fibroblast lines from three different SPG50 patients and heterozygous controls (same sex parent) (Supplemental Table 1). For high content imaging and automated image analysis, high-throughput confocal imaging was performed using the ImageXpress Micro Confocal Screening System (Molecular Devices) using an experimental pipeline described in Behne et al (60) and Ebrahimi-Fakhari et al (32). Dr. Bonifacino's group tested the vector in fibroblast lines from two sibling patients with a donor splice site pathogenic mutation in intron 14 of the AP4M1 gene (c.1137+1G→T) (Patient 1: 87RD38 and Patient 2: 87RD39) (3,33) comparing them to skin fibroblasts from a control individual (85E0344) (see Supplemental Methods and Materials for detailed information).…”

Functional validation of novel variants in B4GALNT1 associated with early‐onset complex hereditary spastic paraplegia with impaired ganglioside synthesis

“…Fibroblasts from the proband and a healthy unrelated control were obtained and cultured as described previously (Behne et al, 2020; Ebrahimi‐Fakhari et al, 2021).…”