Fig 1: 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.