Fig 1: C9orf72 associates with the autophagy initiation complex. (a) Schematic of the LAP-tagged long C9orf72 isoform. (b) Immunoblotting of C9orf72 in an inducible stable cell line expressing a single copy of the long C9orf72 isoform. Lysates from MEFs generated from wild-type and c9orf72 knockout (c9 KO) mice were used to identify the correct immune-reactive band as endogenous C9orf72, as multiple bands were recognized by commercially available C9orf72 antibody. The LAP-tagged C9orf72 long isoform expresses at a comparable level to the endogenous protein. (c) Schematic of SILAC-affinity purification to identify C9orf72-associating proteins. Cell lines stably expressing the LAP-tagged C9orf72 were grown in isotopically ‘heavy’ medium containing 13C6,15N4-arginine and 13C6,15N2-lysine, while the parental line (i.e., no transgene) was grown in ‘light’ medium containing normal arginine and lysine. Elution after the PreScission cleavage step was used for mass spectrometry analysis. (d) Graphic representation of the C9orf72 interactome. Y-axis displays the average ratio of peptides identified in the heavy vs light labeling, while the X-axis represents individual proteins; a protein was considered to be C9orf72-associated if it was at least 8-fold above background signal. The protein ID, median SILAC ratio, and numbers of unique peptides identified are listed. (e) Confirmation of mass spectrometry results by IP-western blots, including RB1CC1, ULK1, ATG13, SMCR8 and WDR41. (f) Reciprocal immunoprecipitation using HA-tagged ATG13 and ATG101. Cells were transfected with plasmids as indicated with pcDNA6 as a control. HA-tagged ATG13 and ATG101 pulled down the LAP-tagged long C9orf72 isoform. (g) Schematic representation of in situ proximity-ligation assay. (h) Representative results for in situ proximity-ligation assay. Phalloidin staining, which labels F-actin, was used to outline the cells. Scale bar: 20 µm. (i) Quantification of in situ PLA results (N = 3, cell numbers >20 per experiment, ***, p < 0.001). C9, C9orf72.
Fig 2: The long, but not the short, isoform of C9orf72, partially rescues the autophagy and the dendritic arborization phenotype. (a) Images of transfected hippocampal neurons from control and c9orf72 knockout mice. Nuclear-localized GFP and membrane-bound mCherry linked by P2A peptide (GFPNLS-P2A-mCherryCAAX, pSYC97), or long or short C9orf72 isoform replacing GFPNLS were examined for their effects on dendritic arborization and autophagy in c9orf72 knockout neurons. Genotypes of neurons are listed in the top panel. The second panels indicate the rescue constructs used: GFPNLS (GFPNLS-P2A-mCherryCAAX, long C9orf72 (long C9ORF72-P2A-mCherryCAAX), and short C9orf72 (short C9ORF72-P2A-mCherryCAAX). The images are presented in gray scale and inverted color. Scale bar: 20 µm. (b,c) Sholl analysis of dendritic arborization (b) and spine density (c) of the rescue experiments (at least 4 independent experiments, N > 3 neurons per genotype per experiment, n.s.: not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001). (d) Fluorescent images of c9orf72 knockout hippocampal neurons transfected with photo-convertible GFP (tdEOS)-tagged LC3 without or with long C9orf72 (long C9ORF72-P2A-mCherryCAAX) or with short C9orf72 (short C9ORF72-P2A-mCherryCAAX) rescue constructs. Insets show a scaled-up image of the boxed region. Scale bar: 10 µm. (e) Quantification of LC3-II puncta in c9orf72 knockout hippocampal neurons with or without the long C9orf72 rescue construct (at least 3 independent experiments N > 3 neurons per genotype per experiment, ***, p < 0.001). (f) LMNB1-immunofluorescent images of hippocampal neurons from control and c9orf72 knockout mice. Nuclear envelopes were apparently normal in both wild-type and c9orf72 knockout hippocampal neurons. Scale bar: 10 µm. (g) Relative mRNA expression levels of C9orf72, Ulk1, Rb1cc1, Atg13, and Atg101 in primary cortical neurons cultured from wild-type and c9orf72 knockout mice. At least 3 independent cultures were used (n.s.: not significant; ***, p < 0.001). (h) Immunoblots of RB1CC1, ATG13, SQSTM1, TARDBP and GAPDH on protein lysates of primary cortical neurons cultured from wild-type and c9orf72 knockout mice.
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