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.

Figure 1

The AP-4 cargo protein ATG9A accumulates in the trans-Golgi network of fibroblasts from patients with AP4B1-associated HSP. (A, B) Western blotting of whole cell lysates of fibroblasts from three patients with bi-allelic variants in AP4B1 reveals a reduction in the levels of AP4E1, a subunit and surrogate for levels of the AP-4 complex. (C) Co-immunoprecipitation of AP4B1 and AP4E1 demonstrates AP-4 complex formation in lysates from heterozygous controls but not in fibroblasts from patients with bi-allelic variants in AP4B1. (D) Whole cell levels of ATG9A by western blotting are significantly increased in patient-derived fibroblasts. (E) Immunocytochemistry for ATG9A (green), trans-Golgi marker TGN46 (red) and nuclear marker DAPI (blue) demonstrates an accumulation of ATG9A in the perinuclear trans-Golgi network in AP-4-deficient patient fibroblasts. Line blots confirm that the ATG9A signal in patient-derived fibroblasts largely overlaps with TGN46 while the signal outside the trans-Golgi network is diminished. Quantification of the area of ATG9A staining overlapping with TGN46 as a ratio to the total area of ATG9A staining confirms the accumulation of ATG9A in the trans-Golgi network of AP-4-deficient fibroblasts. Scale bar: 10 μm (merged); 5 μm (ATG9A perinuclear). A.U., arbitrary units; LoF, loss of function; WT, wild type.

Figure 2

Trafficking of ATG9A is impaired in AP-4-deficient fibroblasts from patients with bi-allelic variants in different AP-4 subunits. (A) Immunocytochemistry for ATG9A (green), trans-Golgi marker TGN46 (red) and nuclear marker DAPI (blue) demonstrates an accumulation of ATG9A in the perinuclear trans-Golgi network in fibroblasts from patients with bi-allelic variants in AP4B1, AP4M1, AP4E1 or AP4S1. (B) In fibroblasts from healthy controls, ATG9A shows an even cytoplasmic distribution. (C) Quantification of the area of ATG9A staining overlapping with TGN46 as a ratio to the total area of ATG9A staining confirms the accumulation of ATG9A in the trans-Golgi network of AP-4-deficient fibroblasts. (D) Western blotting of whole cell lysates from patient fibroblasts with a variety of bi-allelic variants in different AP-4 subunits shows a significant increase in levels of ATG9A and a reduction in levels of AP4E1. The latter serves as a surrogate for levels of the AP-4 complex. Scale bar: 10 μm. LoF, loss of function; WT, wild type.

Figure 3

Re-expression of AP4B1 in fibroblasts from patients with AP4B1-associated HSP leads to a reduction of total ATG9A levels and redistribution from the trans-Golgi network to the cell periphery. (A, B) mRNA expression levels of AP4B1 in wild-type fibroblasts and fibroblasts with heterozygous or bi-allelic loss-of-function variants in AP4B1 with and without treatment with lentivirus to express human AP4B1. (C, D) Re-expression of AP4B1 in AP4B1-deficient patient-derived fibroblasts lowers ATG9A protein levels to levels that are not different from controls. (E) Re-expression of AP4B1 in AP4B1-deficient patient-derived fibroblasts leads to re-distribution of ATG9A from the trans-Golgi network to the cell periphery. Quantification of the area of ATG9A staining overlapping with TGN46 as a ratio to the total area of ATG9A staining demonstrates that the pattern after re-expression of AP4B1 is not significantly different from the pattern in heterozygous controls. Scale bar: 5 μm. Lenti, lentivirus; LoF, loss of function; TGN, trans-Golgi network; WT, wild type.

Figure 4

Autophagic flux is intact in AP-4-deficient patient-derived fibroblasts. (A) Whole cell levels of autophagosome marker LC-3 (as a ratio of LC3-II/LC3-I) and autophagy substrate p62 are similar in fibroblasts with bi-allelic loss-of-function variants in AP4B1 and heterozygous controls under nutrient-rich conditions. (B) When challenged in a paradigm of autophagy induction through starvation and autophagy blockade with bafilomycin A1, AP-4-deficient patient fibroblast shows (C) an increase in LC3II/I indicative of preserved autophagic flux. (D, E) Levels of ATG9A and AP4E1 remain unchanged by autophagy induction or inhibition. (F-H) Immunocytochemistry for LC3-positive autophagosomes and p62 demonstrates a significant increase in vesicle/punctae number and size following autophagy induction and blockade, again arguing that autophagic flux is maintained in AP-4-deficient fibroblasts. Scale bar: 20 μm. BafA1, bafilomycin A1; LoF, loss of function; WT, wild type.

Figure 5

ATG9A is mislocalized in iPSC-derived cortical neurons from AP-4-HSP patients. (A) Fibroblasts from three families with AP4B1-associated HSP were reprogrammed into iPSCs and subsequently into excitatory cortical neurons using overexpression of neurogenin 2. iPSC-derived neurons were grown in 96 well plates and subjected to high-content confocal imaging to assess the localization of ATG9A. Immunocytochemistry was performed for the neuronal marker Tuj-1 (red), ATG9A (green), Golgi marker GM130 (pink) and nuclear marker DAPI (blue). ATG9A concentrated to a high-intensity juxtanuclear area that overlapped with GM130 with lower fluorescence intensity in the remainder of the soma. (B) In neurons from heterozygous controls, ATG9A was distributed in the soma, and the juxtanuclear high-intensity area (marked in yellow), if present, was often small. A significant increase in the high intensity juxtanuclear ATG9A area occurred in patient-derived neurons. The ratio between soma size by Tuj-1 staining (marked in cyan) and the ATG9A high intensity area (marked yellow) was significantly increased in cortical neurons from AP-4-HSP patients compared to controls. This was quantified on (C) a per cell basis with over 90x10³ neurons analyzed per condition or (D) per differentiation with three independent rounds of differentiation per pair of cell lines. (E–I) Western blotting of whole cell lysates of iPSC-derived neurons from three patients with bi-allelic variants in AP4B1 reveals a reduction in the levels of AP4E1, a subunit and surrogate for levels of the AP-4 complex, increased levels of ATG9A and a decrease in the ratio of LC3-II to LC3-I. Scale bar: 20 μm. HI, high intensity; iPSC, induced pluripotent stem cells; LoF, loss of function; NGN2, neurogenin 2; WT, wild type.

Figure 6

Neurite outgrowth is impaired in iPSC-derived cortical neurons from AP-4-HSP patients. (A, B) iPSC-derived cortical neurons from patients with AP4B1-associated HSP and their heterozygous same sex parent were analyzed using automated live cell imaging to assess neurite outgrowth and branching. Neurons were monitored from 4 h post plating until fixation at 24 h. Automated image analysis revealed reduced neurite outgrowth (neurites are pseudo-colored in violet) with a shorter average neurite length and a reduced number of branches per cell (cell bodies are pseudo-colored in orange) in AP4B1-deficient neurons. (C) At 24 h post plating, AP-4-deficient neurons show a robust increase in high-intensity juxtanuclear ATG9A signal (marked in yellow) compared to control, similar to more mature neurons at day 7 post plating (Fig. 5). Scale bar: 100 μm (A); 20 μm (C). HI, high intensity; LoF, loss of function; WT, wild type.