Generation of stable C9orf72-LOF model in zebrafish
To better understand the role of C9orf72-LOF in ALS/FTD pathogenesis, we generated a stable transgenic zebrafish gene-silencing model. A single conserved C9ORF72 ortholog is present in zebrafish on its chromosome 13. To achieve transgenic c9orf72 gene silencing in zebrafish, we used a recent miRNA-based gene-silencing approach developed for zebrafish 25. Unlike morpholino-based knockdown approach, transgenic zebrafish lines that have been constructed to stably express miRNAs designed to target knockdown desired genes of interest have no apparent non-specific toxic effects 26. The miRNA knockdown technique consists in the use of transgenic DNA construct allowing the expression of synthetic miRNA targeting the 3’ UTR of a gene-of-interest, here the endogenous zebrafish c9orf72 (Fig. 1a). As presented more in details in the method section, we designed 4x different miRNAs targeting specifically c9orf72 (C9orf72-miR) that we inserted downstream of a dsRED marker and under the control of a ubiquitous promoter (Ubiquitin), the overall sequence was recombined into a mini-Tol2-R4R2 destination plasmid. To generate a transgenic line, this Tol2-DNA construct was co-injected with transposase mRNA in fertilized eggs at one-cell stage for enhanced genomic integration of the DNA construct 27. To ease the selection of the founders/carriers, we also included an eGFP cassette under the crystallin promoter (Fig. 1b). Founders with eyes displaying GFP fluorescence were selected and raised to generate a stable and heritable C9orf72-miR LOF line (hereafter referred as C9-miR). F1 transgenic fish gave a birth to a ratio of close to 50% positive GFP embryos when outcrossed with wild-type animals, suggesting the presence of a single genomic insertion.
We first analysed C9orf72 silencing efficiency in our C9-miR line by RT-qPCR and western blotting. We showed a significant decrease in the level of C9orf72 mRNA (Fig. 1c) associated with a 50% decrease of C9orf72 protein (Fig. 1d, e). Altogether, these results indicate that our genetic approach efficiently reduces the C9orf72 protein levels in vivo and this C9-miR line can be used to understand the role of C9orf72 haploinsufficiency in ALS.
C9orf72-LOF model shows early motor behavioural defects and reduced viability
We did not observe any overt morphological abnormalities during embryonic development (0–5 dpf) in C9-miR fish (Fig. 2a). From 6–14 dpf, C9-miR larvae exhibited gradual morphological defects such as an unusual body curve and premature death (Fig. 2b,c). C9orf72 partial depletion importantly led to a significant decrease in survival at 10 dpf compared to wild-type controls; with a survival rate of 2–5% after 15 dpf (Fig. 2b).
We, next, examined whether normal zebrafish motor behaviour was affected in larval C9-miR zebrafish (4–11 dpf). To assess motor activity, larval zebrafish that did not display any of the abnormal morphological defects were selected and monitored using the automated Noldus Ethovision XT behaviour monitoring system. A significant decrease in motor activity was observed in C9-miR fish as compared to controls, as of 6 dpf (Fig. 2c,d). Such an impaired motor behaviour early on in C9-miR zebrafish is consistent with findings that we and others have reported in several other zebrafish models of ALS 24,28−30.
C9orf72-LOF zebrafish model display adult hallmark features of ALS
C9-miR fish that survive past 15 dpf were also studied at adult stages for hallmarks of ALS such as muscle atrophy, motoneuron death and paralysis. Hematoxylin & eosin (H&E) staining of cross-section of fish body trunk revealed that muscle in adult C9-miR exhibited severe atrophy (Fig. 3a), with a significant reduction in the thickness of the fibres (Fig. 3b). Choline acetyltransferase (ChAT) staining is a hallmark feature of cholinergic motor neurons. ChAT immunostaining was performed on the spinal cord sections of adult C9-miR fish and the mature motor neurons in the C9-miR fish were reduced in size by 19.2 ± 0.02 % (Fig. 3c). At the motor behavioural level, we observed an impaired swimming ability in C9-miR compared to controls (Fig. 3d, Supplemental Videos). Prior to death, C9-miR fish spent their time in the bottom of the tank with weak movements. Adult survival was also monitored and we observed that by 16 months post-fertilisation, more than 80-90% of the adult C9-miR zebrafish die.
Cytoplasmic aggregation of Trans-activation response element (TAR) DNA-binding protein 43 (TDP-43) is a major pathological hallmark of ALS 31. TDP-43 form aggregates in neurons, glial cells 31 and axial skeletal muscle 32. By taking advantage of the relatively large nucleus and cytoplasm of skeletal muscle cells, we examined whether TDP-43 pathology exist in our model. Using a specific antibody that recognizes the highly homologous human TDP-43 ortholog in zebrafish 33, we showed that this protein is localized to the nucleus of the skeletal muscle cells in wild-type zebrafish (Fig. 4a). In contrast, in C9-miR zebrafish, we observed clusters of TDP-43 in skeletal muscles (Fig. 4b). We then analyzed these clusters further to examine their precise cellular localization and found that they are predominantly located outside of the nucleus. Altogether, our findings provide strong evidences that C9orf72 silencing in zebrafish recapitulates key pathological hallmarks of ALS.
C9orf72 silencing affects NMJ structural integrity and quantal release
We next examined NMJ integrity by performing double-immunohistochemistry on fixed embryos using specific presynaptic (SV2) and postsynaptic markers (a-bungarotoxin). Analysis revealed no change the primary motor neuron axon architecture and in colocalization of pre- and post-synaptic signals in C9-miR fish at 2 dpf (Fig. 5a,b) and 4 dpf. However, in 6 dpf C9-miR larvae, we observed a significant reduction in the number of colocalizing pre- and post- synaptic puncta (Fig. 5c,d). These results indicate that, while the synaptic structures of the NMJ develop properly and are preserved at early embryonic stages in C9-miR, they do start to degenerate from 6 dpf.
To investigate if alterations in NMJ integrity had functional consequences on synaptic transmission in the 6 dpf C9-miR larvae, we recorded and analysed the spontaneous miniature end plate currents (mEPCs) that occur naturally and spontaneously at synapses and represent the unitary event during synaptic transmission (Fig. 6a). We observed that the frequency of mEPCs in C9-miR was significantly reduced compared to controls (Fig. 6b), suggesting a reduction in the number of functional presynaptic endings. The mean amplitude of mEPCs was also found to be smaller in zebrafish C9-miR compared to wild-type zebrafish (Fig. 6c). We observed that the mEPCs from the muscle of C9-miR larvae and controls shared similar rise time and decay time constant kinetics (Fig. 6d).
C9orf72 regulates synaptic vesicle exocytosis and synapse stability at the NMJ
To gain more insights into molecular processes and pathways affected, we determined global changes at the proteomic levels by isolating total proteins at 6 dpf from C9-miR and wild-type siblings. We identified a total of 2602 proteins that were covered by two or more uniques peptides and were quantifiable in four biological replicates (FDR≤1%). Most of the proteins in wild-type and C9-miR were at comparable expression levels. Only 24 proteins were found to be dysregulated (p<0.05; Table S2). Of these hits, 12 were upregulated and 12 were downregulated in C9-miR fish (Fig. 7a). These differentially expressed proteins (DEPs) were classified into functional clusters according to the PANTHER classification system (Fig. 7b-e). The classification results revealed that many DEPs were distributed into six protein classes (Fig. 7b). These proteins are classified in three molecular functions namely binding (20%), structural molecule activity (20%) and catalytic activity (60%) (Fig. 7c). They are involved in biological processes, being cellular process, metabolic process and biological regulations the most represented ones with 38%, 23.1% and 15.4% of proteins respectively (Fig. 7d). Cellular component analysis revealed that the DEPs belong in majority to the organelle, membrane and synapse categories (Fig. 7e). Consistent with the synaptic dysfunction phenotype, we identified a strong downregulation of synaptic proteins (Fig. 7a; Table S2). Among these proteins, the top hit of dysregulated proteins is the synaptic protein, synaptic vesicle-associated protein 2a (SV2a). Importantly, a recent study showed that SV2a is reduced in C9orf72-ALS patient-derived IPS neurons 34. This data links the findings in our C9orf72 loss-of-function model to ALS.
Given that SV2a is an essential component of active zones and synaptic release machinery, we next sought to further assess synaptic activity at the NMJ by measuring synaptic vesicle (SV) cycling at the NMJ in zebrafish larvae using the fluorescent styryl dye, FM1-43 35,36. C9-miR and controls larvae were exposed to FM1-43 and its uptake into NMJ presynaptic boutons was monitored. The presynaptic terminals were acutely depolarized with a high [K+] HBSS solution (45 mM) to drive the exocytotic activity, SV cycle and load FM1-43 and label synaptic clusters. In controls, we observed strong fluorescence staining along terminal axon branches at individual synaptic varicosity boutons (Fig. 8a). While in C9-miR fish we found a significant reduction in FM1-43 loading in presynaptic terminals (Fig. 8b), indicating slowing of the exocytotic activity and the overall SV cycle. These findings reveal a key role for C9orf72 in regulating presynaptic vesicle release at NMJ.
To assess organization of the presynaptic structure at NMJ, we examined the expression of Rab3a, a protein associated with vesicles at active zones that is essential for synaptic vesicle release and neurotransmission (Fig. 8c-e). We found a reduced number of Rab3+ puncta in C9-miR fish compared to controls (Fig. 8c-d) as well as the area of the putative synapses were smaller in C9-miR fish (Fig. 8e).