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The mouse C9ORF72 ortholog is enriched in neurons known to degenerate in ALS and FTD

Abstract

Using transgenic mice harboring a targeted LacZ insertion, we studied the expression pattern of the C9ORF72 mouse ortholog (3110043O21Rik). Unlike most genes that are mutated in amyotrophic lateral sclerosis (ALS), which are ubiquitously expressed, the C9ORF72 ortholog was most highly transcribed in the neuronal populations that are sensitive to degeneration in ALS and frontotemporal dementia. Thus, our results provide a potential explanation for the cell type specificity of neuronal degeneration caused by C9ORF72 mutations.

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Figure 1: Mouse 3110043O21Rik is the ortholog of human C9ORF72.
Figure 2: Distribution of X-gal in heterozygous mice with 3110043O21Rik-LacZ insertions.
Figure 3: Characterization of the cells expressing β-gal under the control of the 3110043O21Rik promoter.

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Acknowledgements

The authors wish to thank all members of the Eggan laboratory for their technical support and helpful discussions. We also thank the Harvard Stem Cell and Regenerative Biology Histology Core. This work was supported by the Howard Hughes Medical Institute, Project ALS, p2ALS, Target ALS and National Institute of Neurological Disorders and Stroke grant #164520. N.S. was supported by the 2011 Lilly Scientific Fellowship Program.

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Authors and Affiliations

Authors

Contributions

K.E. conceived the project. K.E., N.S., A.M.M. and F.T.M. designed the experiments. F.T.M. performed in situ hybridization. K.K. supported the animal experiments. A.M.M., A.I., I.A. and N.S. supported staining. R.M. and B.N.D.-D. supported RNA sequencing data analysis. N.S., A.M.M. and K.E. wrote the manuscript. K.E. supervised the project.

Corresponding author

Correspondence to Kevin Eggan.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gene tree of C9ORF72 gene and its orthologs.

Note that C9ORF72 orthologs are highly conserved among several species, especially in vertebrata. Black line represents x1 branch length, while blue line represents x10 branch length. Green box represents alignment match, while white box represents alignment gap. F18A1.6 in C elegans has 23 % identity with human C9ORF72.

Supplementary Figure 2 Amino acid sequence of human C9ORF72 and mouse 3110043O21Rik gene.

Upper and lower lane represent human and mouse ortholog respectively.

Supplementary Figure 3 Comparison of splicing isoforms between human C9ORF72 (a), mouse 3110043O21Rik gene (b).

Black boxes represent translated exons and grey boxes represent predicted non-coding exons. Blue font represents translated isoforms. Red arrowhead and dashed line represent the location of hexanucleotide repeat expansion in human patients with ALS/FTD (a) and analogous site of patient C9ORF72 hexanucleotide repeat (b). RNA sequence data (c-e). Isoform 1 is highly expressed in mouse cortex (e). FPKM, Fragments Per Kilobase of exon per Million mapped fragments. Error bars indicate s.d. n=2 for (c), n=3 for (d) and (e).

Supplementary Figure 4 Comparison of sequence between human C9ORF72, mouse 3110043O21Rik and chimpanzee LOC465031 genes around the sequence of hexanucleotide repeats in human.

58.3 % identities are found between human and mouse. Note that GC rich region in both sequences.

Supplementary Figure 5 X-gal staining in the brain and several organs of C9ORF72-ortholog knockin mice.

(a) 0.6 mm from the midline. A, anterior; P, posterior; OB, olfactory bulb; cc, corpus callosum; CPu, caudate putamen (striatum); DC, granular dentate gyrus; thal, thalamus; cereb, cerebellum. Lung (b), liver (c), and kidney (d) are negative for X-gal staining. Testis (e) is shown as positive control. Bar, 1 mm (a) and 50 μm (b-e).

Supplementary Figure 6 Co-localization of β-gal, Cux1, and NeuN is shown (a).

β-gal positive cells are also located in superficial layers (b). (b) shows Z-stack and orthogonal images of white rectangle in (a). Bar, 50 μm (a) and 20 μm (b).

Supplementary Figure 7 Co-localization of β-gal, ChAT, and NeuN in ventral horn of spinal cord.

(b-d) shows Z-stack and orthogonal images of white rectangle in (a). Arrowheads show ChAT positive motor neurons and arrows are ChAT negative cells. Bar, 50 μm (a) and 20 μm (b-d).

Supplementary Figure 8 β-gal positive cell does not co-localize with IbaI and GFAP in anterior horn of spinal cord (a-f).

Images are taken from the same slice. Z-stack images are also shown. Green-β –gal, red-IbaI (e) or GFAP (f) and blue-DAPI. Bar, 50 μm (a-f).

Supplementary Figure 9 Brain expression pattern of C9ORF72 ortholog from Allen Brain Atlas.

(a-l) Expression pattern of C9ORF72-ortholog of brain from Allen Brain Atlas database. Allen Brain Atlas used probes from exon 4 through exon 11. In situ hybridization of C9ORF72-ortholog in sagittal section of mice brain at P56: Nissl staining (a) and expression image (b) from similar slices. Higher magnification of cortex (c, d), hippocampus (e, f), brainstem (g, h), cerebellum (i, j), and thalamus (k, l) from similar slices. Nissl staining (a, c, e, g, i, k) and expression of C9ORF72-ortholog (b, d, f, h, j, l). Note that hippocampus has higher expression. Bar, 1 mm (a, b) and 300 μm (c-l). Note that each image is a distinct section.

Supplementary Figure 10 Spinal cord expression pattern of C9ORF72 ortholog from Allen Brain Atlas.

(a-g) Expression pattern of C9ORF72-ortholog in spinal cord from Allen Brain Atlas database. Allen Brain Atlas used probes from exon 4 through exon 11. In situ hybridization of C9ORF72-ortholog in sagittal section of mouse brain at P56: Nissl staining (a) and expression image (b-g) from similar slices. Lower (a-d) and higher (e-g) magnification are presented. Expression pattern of C9ORF72-ortholog (d, e), ChAT (b, f) and GFAP (c, g) are shown. Note that C9ORF72-ortholog transcript distributes mainly in the grey matter and displays a pattern similar to, but modestly broader than ChAT. White line shows the border between grey and white matter (e-g). Bar 200 μm (a-d), 100μm (e-g). Note that each image is from a distinct section.

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Supplementary Figures 1–10, Supplementary Tables 1–2 (PDF 5180 kb)

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Suzuki, N., Maroof, A., Merkle, F. et al. The mouse C9ORF72 ortholog is enriched in neurons known to degenerate in ALS and FTD. Nat Neurosci 16, 1725–1727 (2013). https://doi.org/10.1038/nn.3566

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