Reports / http://www.sciencemag.org/content/early/recent / 7 February 2013 / Page 1/ 10.1126/science.1232927 Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are the extreme ends of a spectrum of overlapping neu- rodegenerative disorders variably associated with dementia, personality changes, language abnormalities and progressive muscle weakness (1– 3). The majority of patients show intracellular inclusions that are strong- ly positive for phosphorylated TDP-43 (classified as FTLD-TDP, FTLD/ALS-TDP or ALS-TDP). Recently, expansion of a GGGGCC hexanucleotide repeat in the gene C9orf72 has been identified as the most common pathogenic mutation in families with autosomal dominant FTLD, FTLD/ALS and ALS (4–6). The expansion is located upstream of the C9orf72 coding region, either in the first intron or the promoter region, depending on the transcript isoform (fig. S1A). Although the extreme GC-content precludes sequencing in patients, the number of GGGGCC repeat units is believed to be at least several hundred com- pared to fewer than 25 in healthy controls (7). Patients with a C9orf72 repeat expansion mutation have clinical symptoms similar to other FTLD/ALS-TDP patients, but show several unique pathological features (8–12). Aggregates of phosphorylated TDP- 43 are accompanied by abundant dot-like and star-shaped phospho-TDP- 43-negative neuronal cytoplasmic inclusions in particular in cerebellum, hippocampus and frontotemporal neocortex that can only be identified with antibodies for p62, ubiquitin or the related ubiquilins (8–11). These phospho-TDP-43 negative aggregates are highly characteristic of dis- eased C9orf72 mutation carriers and are absent in other variants of FTLD/ALS-TDP (9–11). The identity of the disease protein(s) in these inclusions and their relation to the C9orf72 hexanucleotide repeat expan- sion has remained elusive. Proposed pathomechanisms include haploinsuffi- ciency through impaired transcription or splicing of the mutant C9orf72 allele and RNA toxicity through the seques- tration of unidentified RNA-binding proteins (3–7). We hypothesized that the intronic GGGGCC-repeat might be aberrantly translated into dipeptide-repeat (DPR) proteins. Although quite rare, two me- chanisms of non-ATG-initiated transla- tion have been described: Initiation from alternative start codons with a good Kozak sequence is possible (13– 15); and hairpin-formation in the repeat region may trigger so-called repeat- associated non-ATG-initiated (RAN) translation, as described for CAG- repeats in ataxin 8 (ATXN8) (16–18). ATXN8 encodes a natural poly-Q stretch that can cause poly-Q inclusions upon repeat expansion in spinocerebel- lar ataxia type 8 (SCA8) patients. Stri- kingly, the expanded CAG-repeat is translated in all three reading frames (poly-Q, poly-A and poly-S) even after removal of the endogenous start codon. Translation of the GGGGCC-repeat in all reading frames would result in three dipeptide-repeat (DPR) proteins poly-(Gly-Ala), poly-(Gly-Pro) and poly-(Gly-Arg). poly-GA and poly-GP proteins are extremely hydrophobic and may form intracellular aggregates. We raised antibodies (anti-GA and anti-GP) against (GA) 15 and (GP) 15 peptides fused to maltose binding protein and tested a monoclonal antibody (anti- GR) that was originally raised against an EBNA2A epitope with a (GR) 6 repeat (19). All three affinity-purified DPR antibodies detected the respective repeat antigen by immunoblotting without cross-reaction with the other two DPR proteins (Fig. 1A). To test whether such repeat proteins can be translated in the absence of a start codon, we cloned parts of the repeat region from C9orf72 pa- tients into a mammalian expression vector. For the longer constructs we could only use restriction digest to estimate the repeat number ranging from ~28 to ~145 (Fig. 1B), because the extreme GC-content precludes sequencing. The region upstream of the GGGGCC-repeat lacks ATG start codons and contains four to five stop codons depending on the read- ing frame. Upon transfection of these constructs into HEK293 cells, anti- GA antibodies detected proteins of increasing size starting with a faint product from ~38 repeats, suggesting that the translation mechanism becomes more efficient with increasing repeat length (Fig. 1B). We did not detect poly-GR products and only the longest construct with ~145 repeats additionally expressed detectable amounts of poly-GP (Fig. 1B). To analyze poly-GA aggregation we performed filter trap assays (20) using 2% sodium dodecyl sulfate (SDS) extracts from human post- mortem cerebellum of healthy controls and FTLD/ALS-TDP patients with and without pathological C9orf72 hexanucleotide repeat expansion. We observed strong poly-GA signal only in FTLD/ALS patient with hexanucleotide repeat expansion (Fig. 1C) indicating that poly-GA forms SDS-insoluble aggregates in the cerebellum. We also detected insoluble poly-GP and poly-GR in C9orf72 patients (Fig. 1C). The 2% The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide- Repeat Proteins in FTLD/ALS Kohji Mori, 1 * Shih‐Ming Weng, 2 * Thomas Arzberger, 3 Stephanie May, 2 Kristin Rentzsch, 2 Elisabeth Kremmer, 4 Bettina Schmid, 2,5 Hans A. Kretzschmar, 3 Marc Cruts, 6,7 Christine Van Broeckhoven, 6,7 Christian Haass, 1,2,5 Dieter Edbauer 1,2,5 † 1 Adolf Butenandt Institute, Biochemistry, Ludwig-Maximilians University Munich, Schillerstrasse 44, 80336 Munich, Germany. 2 German Center for Neurodegenerative Diseases (DZNE), Munich, Schillerstrasse 44, 80336 Munich, Germany. 3 Center for Neuropathology and Prion Research, Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 23, 81377 Munich, Germany. 4 Institute of Molecular Immunology, Helmholtz Zentrum München, Marchioninistrasse 25, 81377 Munich, Germany. 5 Munich Cluster of Systems Neurology (SyNergy), Ludwig-Maximilians-University Munich, Schillerstrasse 44, 80336 Munich, Germany. 6 Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Universiteitsplein 1, 2610 Antwerp, Belgium. 7 Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium. *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: dieter.edbauer@dzne.de Expansion of a GGGGCC hexanucleotide repeat upstream of the C9orf72 coding region is the most common cause of familial frontotemporal lobar degeneration and amyotrophic lateral sclerosis (FTLD/ALS), but the pathomechanisms involved are unknown. Like in other FTLD/ALS variants, characteristic intracellular inclusions of misfolded proteins define C9orf72 pathology, but the core proteins of the majority of inclusions are still unknown. Here we found that most of these characteristic inclusions contain poly-(Gly-Ala) and to a lesser extent poly-(Gly-Pro) and poly-(Gly- Arg) dipeptide-repeat proteins presumably generated by non-ATG-initiated translation from the expanded GGGGCC repeat in three reading frames. These findings directly link the FTLD/ALS-associated genetic mutation to the predominant pathology in patients with C9orf72 hexanucleotide expansion. on February 9, 2013 www.sciencemag.org Downloaded from