Fig 2: Schematic representation of C9orf72 gene structure, RNA, and protein products, and the strategy for detecting C9orf72 transcripts.a C9orf72 genomic structure is illustrated; boxes represent exons. Exon 1a and 1b (navy), which do not encode protein, are alternative first exons, and exon 2 contains the translational start site (ATG, black triangle). The G4C2-repeat expansion (aqua) is in intron 1, upstream of exon 1b. Approximate position of primers used to detect various transcripts in Taqman assays are illustrated with arrows, including primers that span the exon 2–exon 3 junction (black) that detects all transcripts, primers that span the exon 1a–exon 2 junction (coral) that are specific to V3, and primers complementary to intron 1 (aqua) that detect all intron 1-containing and expansion-containing variants. b pre-mRNAs corresponding to V1–V3 are illustrated. The coral star indicates SS1b. c Mature mRNAs for all variants and the proteins they encode are shown. Primers for all variants detect all three mature mRNAs, whereas the V3 primers are specific to V3. Intron 1 primers do not detect any mature mRNAs. d Pathogenic RNA by-products resulting from the G4C2-repeat expansion in intron 1 are shown. The location of the G4C2-repeat expansion (aqua) is shown in sense and antisense RNAs. Intron 1 primers detect all pathological RNAs, and the all variants primers will detect any mis-spliced V1/V3 transcripts that include exon 2. SS1b Splice site 1b, V variant.

Fig 3: Loss of C9orf72 reduces levels of synapsin in the hippocampus in vivo. Representative images of hippocampal sections of 12-week-old C9orf72-WT, C9orf72-HET and C9orf72-KO mice stained for a Syn1, b Syn3 or c SV2. Syn1, Syn3, and SV2 levels in the mossy fibre area (outlined) were quantified as the area fraction of positive staining (Area Fraction) and the mean fluorescence intensity level (Intensity) within the outlined area was determined per section. Scale bar 100 µm. Data are presented as box and whisker plots; a Syn1, n (sections analysed) WT = 26, HET = 26, KO = 27 from 5 animals/genotype; b Syn3, n (sections analysed) WT = 30, HET = 28, KO = 26 from 5 animals/genotype; c SV2, n (sections analysed) WT = 26 HET = 24 and KO = 16 from 5 animals/genotype. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test, ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 4: Loss of C9orf72 affects synaptic vesicle pools in the hippocampus in vivo. a Representative transmission electron micrographs of the CA3 region of the hippocampus from 12-week old C9orf72-WT or C9orf72-KO mice. Scale bar 0.5 µm. Box denotes zoomed area. An excitatory nerve terminal is outlined by the red line in the zoomed area. Scale bar 0.15 µm. b Quantification of the density of synaptic vesicles (SV density, µm2) and the number of docked synaptic vesicles per synapse (Docked SVs/synapse) in C9orf72-WT and -KO excitatory synapses. Data are is presented as box and whisker plots; n (synapses analysed) WT = 81, KO = 105 obtained from 4 C9orf72-WT and 5 C9orf72-KO animals. Statistical significance was determined by unpaired two-tailed t test, **P < 0.01, ****P < 0.0001

Fig 5: C9orf72 haploinsufficiency affects excitatory neurotransmission and network function. a–c Primary rat hippocampal neurons were transduced with non-targeting control miRNA (miRNA NTC) or C9orf72 miRNA (miRNA C9orf72) lentivirus at 5DIV and miniature excitatory postsynaptic current (mEPSC) traces were recorded from single neurons on 12–13DIV at a holding potential of –70 mV (–84 mV with liquid junction potential correction). a Representative current traces, b mEPSC interevent interval time and c mEPSC amplitude data are presented as cumulative probability and mean ± SEM of n = number of cells recorded n (cells) miRNA-NTC = 15, miRNA-C9 = 13 from three individual batches of neurons. Statistical significance was determined by unpaired two-tailed t test, ns (not significant), *P < 0.05. d–g Multi-electrode array (MEA) recordings to measure network activity were performed at 12–13DIV on hippocampal neuron cultures transduced with non-targeting control miRNA (miRNA-NTC) or C9orf72 miRNA (miRNA-C9) lentivirus at 5DIV. d Representative traces of a single array channel electrode recorded from miRNA NTC or miRNA C9-transduced neurons. Scale bars 5 s, 50 µV. Network activity characteristics were quantified by determining the e interburst interval of network bursts, f intra-network burst spiking frequency, g network burst length. Data are presented as mean ± SEM of n = number of MEA arrays, n (arrays) miRNA-NTC = 6, miRNA-C9 = 7 from 4 individual batches of neurons. Statistical significance was determined by unpaired two-tailed t test, ns (not significant), *P < 0.05