The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway

Abstract

Hexanucleotide repeat expansion in the C9orf72 gene is a leading cause of frontotemporal lobar degeneration (FTLD) with amyotrophic lateral sclerosis (ALS). Reduced expression of C9orf72 has been proposed as a possible disease mechanism. However, the cellular function of C9orf72 remains to be characterized. Here we report the identification of two binding partners of C9orf72: SMCR8 and WDR41. We show that WDR41 interacts with the C9orf72/SMCR8 heterodimer and WDR41 is tightly associated with the Golgi complex. We further demonstrate that C9orf72/SMCR8/WDR41 associates with the FIP200/Ulk1 complex, which is essential for autophagy initiation. C9orf72 deficient mice, generated using the CRISPR/Cas9 system, show severe inflammation in multiple organs, including lymph node, spleen and liver. Lymph node enlargement and severe splenomegaly are accompanied with macrophage infiltration. Increased levels of autophagy and lysosomal proteins and autophagy defects were detected in both the spleen and liver of C9orf72 deficient mice, supporting an in vivo role of C9orf72 in regulating the autophagy/lysosome pathway. In summary, our study elucidates potential physiological functions of C9orf72 and disease mechanisms of ALS/FTLD.

Keywords: Amyotrophic lateral sclerosis; Autophagy; C9orf72; FIP200/RB1CC1; Frontotemporal lobar degeneration; Lysosome; SMCR8; Ulk1; WDR41.

Fig. 1

SILAC proteomic screen for C9orf72 binding partners. a Schematic workflow of SILAC proteomic screen used to identify C9orf72 protein interactions. b Volcano plot of SILAC hits. Hits with more than 10 peptides are plotted. Top hits identified in the heavy fraction are highlighted

Fig. 2

Co-immunoprecipitation between C9orf72, SMCR8 and WDR41. a GFP-tagged human C9orf72 isoform I (GFP-C9-L) or isoform II (GFP-C9-S) were overexpressed with SMCR8-myc in HEK293T cells and immunoprecipitated by anti-GFP beads. b SMCR8-GFP and WDR41-myc were coexpressed with or without FLAG-C9-L and the lysates were immunoprecipitated using anti-GFP antibodies. c GFP-C9-L and FLAG-WDR41 were co-expressed with or without SMCR8-myc as indicated and the lysates were immunoprecipitated using anti-GFP antibodies

Fig. 3

Cellular localization of C9orf72, SMCR8 and WDR41. a HeLa cells were transfected with FLAG-C9orf72 (C9-L), SMCR8-myc or WDR41-GFP. Cells were stained with anti-FLAG or anti-myc to visualize FLAG-C9orf72 or SMCR8-myc, respectively. Maximum projection images from confocal sections are shown. Scale bar = 10 μm. b HeLa cells were transfected with GFP-C9orf72 (C9-L) and SMCR8-myc. Cells were stained with anti-myc antibodies to visualize SMCR8-myc. c WDR41-GFP expressing HeLa cells were treated with DMSO control, 0.3 mM BrefeldinA (BFA), or 20 mM Nocodazole for 2 h. Cells were stained with anti-GPP130 antibodies to label cis-Golgi. Single confocal images are shown for b and c. Scale bar = 10 μm

Fig. 4

The C9orf72/SMCR8/WDR41 complex interacts with FIP200/Ulk1. a Co-immunoprecipitation between C9orf72/SMCR8/WDR41 and FIP200 and Ulk1. HEK293T cells were transfected with FLAG-FIP200 and C9orf72, SMCR8 and/or WDR41 as indicated. Cells were lysed 40 h after transfection and lysates were immunoprecipated with anti-GFP antibodies. Lysates and immunoprecipitates were analyzed by Western blots as indicated. * indicated non-specific bands recognized by anti SMCR8 antibodies in the IP products. Representative images from 3 independent experiments are shown. b Co-immunoprecipitation between C9orf72/SMCR8/WDR41 and FIP200. HEK293T cells were transfected as indicated and lysed and immunoprecipitated using anti-FLAG antibodies. Lysates and immunoprecipitates were analyzed by Western blots. c, d Co-immunoprecipitation between C9orf72/SMCR8/WDR41 and ATG101 (c) or ATG13 (d). HEK293T cells were transfected as indicated and lysed and immunoprecipitated using anti-GFP antibodies. Lysates and immunoprecipitates were analyzed by Western blots. e Schematic drawing of the interaction between C9orf72/SMCR8/WDR41 and the FIP200/Ulk1 complex. WDR41 interacts with the C9orf72/SMCR8 dimer to form ternary complex, which then interacts with the FIP200/Ulk1/ATG13/ATG101 complex

Fig. 5

Generation of C9orf72 deficient mice. a Schematic drawing of the mouse homologue of C9orf72, 3110043O21RIK, and the site targeted for editing by CRISPR/Cas9. b Sequencing traces of wildtype (top) and edited (bottom) C9orf72 from genomic PCR show a one nucleotide deletion (highlighted with yellow) near the Cas9 cleavage site. c Western blot analysis of C9orf72 protein levels in wild type (WT) and C9orf72-/- (KO) mouse tissues with anti-C9-L antibodies. d Representative images of cervical lymph nodes and spleen from 4-5 months old WT and C9orf72-/- mice. Scale bar = 2 mm (lymph node); 1 cm (spleen)

Fig. 6

C9 deficiency in mice results in an enlarged spleen phenotype and macrophage infiltration into the spleen. a H&E staining of spleen tissues from 5 month old of WT or C9orf72 -/- mouse with zoomed images of germinal center (GC) and red pulp (RP). (GC): Arrows indicate plasma cells and the arrowheads indicate immature immune cells). (RP): arrowheads indicate myeloid precursors; arrows indicate erythroid precursors. Scale bar = 500 μm (100 μm in the zoomed in images for GC and RP). b Immunostaining of 4 month old spleen sections (red pulp region) of WT and C9orf72-/- mice with anti-mouse CD68, prosaposin (PSAP), and progranulin (PGRN) antibodies. Nuclei are labelled with DAPI. Insert shows representative CD68+ macrophage cells. Representative pictures from three pairs of mice are shown. Scale bar = 40 μm. c Immunostaining of 4 month old spleen sections (red pulp region) of WT and C9orf72-/- mice with anti-mouse Lamp1, and cathepsin D (CathD) antibodies. Nuclei are labelled with DAPI. Insert shows representative cells. Representative pictures from three pairs of mice are shown. Scale bar = 40 μm

Fig. 7

Increased macrophage infiltration and lysosomal proteins in the cervical lymph node of C9 deficient mice. a Immunostaining of 4 month old cervical lymph nodes sections (peripheral region) of WT and C9orf72-/- mice with anti-mouse CD68, prosaposin (PSAP), and progranulin (PGRN) antibodies. Nuclei is labelled with DAPI. Insert shows representative CD68+ macrophage cells. Representative pictures from three pairs of mice are shown. Scale bar = 40 μm. b Immunostaining of 4 month old cervical lymph nodes sections (peripheral region) of WT and C9orf72-/- mice with anti-mouse Lamp1, and cathepsin D (CathD) antibodies. Nuclei are labelled with DAPI. Insert shows representative cells. Representative pictures from three pairs of mice are shown. Scale bar = 40 μm

Fig. 8

C9 deficiency in mice results in macrophage infiltration and increased levels of lysosomal proteins in the liver. a H&E staining of liver tissues from 10 month old of WT or C9orf72 -/- mouse. (ii) and (iii) are high power magnification images of the hepatic parenchyma, arrowhead indicates infiltrated immune cells; arrow points to necrotic hepatocyte. (v) and (vi) are high power magnification images of the hepatic portal area, arrowhead indicates infiltrated immune cells. PV, portal vein. Scale bar: 500 μm in (i) and (iv), 100 μm in (ii, iii, v, vi) b Immunostaining of 4 month old liver sections of WT and C9orf72-/- mice with anti-mouse CD68, prosaposin (PSAP), and progranulin (PGRN) antibodies. Nuclei are labelled with DAPI. Insert shows representative CD68+ macrophage cells. Representative pictures from three pairs of mice are shown. Scale bar = 40 μm. c Immunostaining of 4 month old liver sections of WT and C9orf72-/- mice with anti-mouse Lamp1, and cathepsin D (CathD) antibodies. Nuclei are labelled with DAPI. Insert shows representative cells. Representative pictures from three pairs of mice are shown. Scale bar = 40 μm

Fig. 9

Microglia do not show any obvious abnormalities in C9orf72 deficient mice. Brain sections from 10 month old WT and C9orf72-/- mice were stained with anti-LAMP1, Iba1 and cathepsin D antibodies. Similar results were seen with 5 month old C9orf72-/- mouse. Scale bar = 50 μm

Fig. 10

C9 deficiency in mice leads to increased levels of autophagy-lysosome proteins in the spleen. a Western blot analysis of tissue lysates from spleen, liver, and brain of WT and knockout mice. Representative pairs are shown. b Quantification of autophagy and lysosomal protein levels in knockout mice relative to WT controls. Data are presented as Mean ± SEM, n = 3–6. *, p < 0.05; **, p < 0.01