Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking

Abstract

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3–5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

AP-4 mediates vesicular transport of the 2-AG endocannabinoid producing enzyme DAGLB

“…Collectively, these observations confirm DAGLB as an AP-4 cargo in HeLa and SH-SY5Y cells, and suggest that TGN export of DAGLB is a ubiquitous function of the AP-4 pathway. AP-4-deficiency increases the expression level of ATG9A in neurons (De Pace et al, 2018;Ivankovic et al, 2020) and patient fibroblasts, and this serves a robust diagnostic disease marker (Behne et al, 2020). Likewise, we observed increased levels of DAGLB in whole cell lysates from AP-4-depleted neuronally differentiated SH-SY5Y cells ( Fig.…”

“…In the absence of AP-4, ATG9A accumulates at the TGN of diverse cell types, including neuronal and nonneuronal cells (Behne et al, 2020;Davies et al, 2018;De Pace et al, 2018;Ivankovic et al, 2020;Mattera et Davies et al, 2020. To assess whether DAGLB exhibits a similar missorting phenotype in AP-4-deficient cells, we used immunofluorescence microscopy.…”

Loss of ap4s1 in zebrafish leads to neurodevelopmental defects resembling spastic paraplegia 52

“…SPG52 is caused by bi‐allelic loss‐of‐function variants in AP4S1 , which encodes the sigma subunit of the adaptor protein complex 4 (AP‐4), and has been described in less than 10 individuals, thus far 3 . AP‐4 role in endosome membrane trafficking was evaluated in a previous study 4 …”

Integrating structural and evolutionary data to interpret variation and pathogenicity in adapter protein complex 4

“…Sivley et al 34,35 developed a program called PathProx that evaluates the relative 3D proximity of a variant to known pathogenic and benign variants. To explore the spatial distribution of variants in AP‐4, we mapped onto the AP‐4 homology model four reported pathogenic variants (curated from the literature) 13–16,36 and six variants annotated as likely pathogenic in ClinVar (Table 3). 32 We also identified 699 likely benign variants in AP‐4 from gnomAD database (Figure 4).…”

“…This is partially because AP‐4 is endogenously expressed at much lower levels than other adaptor proteins, and many model organisms (yeast, flies, worms) lack AP‐4 2 . Genetic variation in AP‐4 has been associated with rare spastic paraplegias and other neurological disorders, 36 but the association of both rare and common genetic variation in AP‐4 with disease has not been comprehensively investigated. To address the need for approaches to interpret new AP‐4 variants, we constructed a homology‐based structural model of the AP‐4 complex; mapped conservation and key AP‐4 variants into this model; and quantified the evolutionary history and structural location of known and predicted variants.…”