Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model

Abstract

Large expansions of hexanucleotide GGGGCC (G4C2) repeats (hundreds to thousands) in the first intron of the chromosome 9 open reading frame 72 (C9orf72) locus are the strongest known genetic factor associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Different hypotheses exist about the underlying disease mechanism including loss of function by haploinsufficiency, toxicity arising as a result of RNA or dipeptide repeats (DPRs). Five different DPRs are produced by repeat-associated non-ATG-initiated translation of the G4C2 repeats. Though earlier studies have indicated toxicity of the DPRs in worms, flies, primary cultured cells and cell lines, the effect of expressing DPRs of amyotrophic lateral sclerosis-relevant length has not been tested on motor behaviour in vertebrate models. In this study, by expressing constructs with alternate codons encoding different lengths of each DPR (40, 200 and 1000) in the vertebrate zebrafish model, the GR DPR was found to lead to the greatest developmental lethality and morphological defects, and GA, the least. However, expressing 1000 repeats of any DPR, including the 'non-toxic' GA DPR led to locomotor defects. Based on these observations, a transgenic line stably expressing 100 GR repeats was generated to allow specific regional and temporal expression of GR repeats in vivo. Expression of GR DPRs ubiquitously resulted in severe morphological defects and reduced swimming. However, when expressed specifically in motor neurons, the developmental defects were significantly reduced, but the swimming phenotype persisted, suggesting that GR DPRs have a toxic effect on motor neuron function. This was validated by the reduction in motor neuron length even in already formed motor neurons when GR was expressed in these. Hence, the expression of C9orf72-associated DPRs can cause significant motor deficits in vertebrates.

Figure 1.

Expression of DPRs of physiologically relevant length has differential effects of survival and morphology. (A) Scheme showing the different constructs used for transient expression. GA, PA, GR and PR DPRs of different lengths (40, 200 and 1000) with C-terminal GFP were used for the toxicity analyses. (B) Different types of defects were observed in the injected larvae at 48 hpf, ranging from slight edema (mild defects) to severe pericardiac edema (severe defects). (C) Quantification of the number of normal, slightly defective, severely defective and dead embryos at 30 hpf after injecting different constructs showed that GA was the least toxic DPR, and GR was the most toxic (N = 3, n = 100 embryos per construct).

Figure 2.

DPR expression impairs locomotor activity. (A) Examples of 5 superimposed swimming paths of zebrafish larvae expressing empty vector (control) or 40, 200 or 1000 repeats of different DPRs tested for touch-evoked escape response. While the expression of 1000 DPRs uniformly resulted in impaired touch-evoked escape responses, expression of 200 DPRs showed variability among the dipeptides. (B) Quantification of the mean distance swam upon a light touch on the tail (N = 2, n = 15 larvae per construct). Expression of 1000 DPRs resulted in impaired swimming in all treatment groups including the GA construct. (Statistical significance was estimated by one-way ANOVA was performed for the different groups: GA: P < 0.0001, GR: P < 0.0001, PA: P < 0.0001, PR: P = 0.003.)

Figure 3.

Stable expression of GR repeats results in abnormal development. (A) Schematic representation of the construct used for generation of the transgenic zebrafish. (B) Schematic showing the cross between the UAS transgenic line and Gal4 driver line. Gal4 driver lines were used to trigger expression of the construct ubiquitously or in motor neurons specifically (using Gal4 under a ubiquitous Hsp, motor neuron-specific Hb9 or pan-neuronal HuC promoter). (C) Western blotting analysis of lysates from 3 dpf transgenic larvae from two transgenic founder lines (GR100-1 and -2) crossed with a Hsp: Gal4 driver subjected to 1 h heat shock at 48 hpf confirms the expression of the GR DPR. Transgenic embryos not subjected to heat shock were used as control. α-tubulin was used as loading control. (D) Proportion of larvae which were morphologically normal, slightly defective, severely defective or dead upon ubiquitous expression of GR100 (left panel; Hsp: Gal4 driver) or motor neuron specific expression of GR100 (right panel; Hb9-Gal4 driver) at 72 hpf. For the heat shock Gal4 expression, heat shock was performed at 37°C at 48 hpf (N = 2, n = 100 larvae per condition).

Figure 4.

Expression of GR DPRs results in locomotor defects. (A) Swimming activity assessed at 7 dpf ubiquitously shows a reduction in swimming activity in transgenic larvae expressing GR DPR (with distance swam by the control in mm normalized to 100%). Non-transgenic larvae subjected to heat shock (negative control) and transgenic larvae not subjected to heat shock were used as controls (N = 2, n = 8 larvae per condition, two-tailed P < 0.0001 calculated by unpaired t-test). Larvae subjected to heat shock are represented using black bars. (B) Swimming behaviour assessed at 7 dpf was observed to be reduced in transgenic larvae expressing GR DPRs specifically in motor neurons. Non-transgenic larvae were used as controls (N = 3, n = 8 larvae per condition, two-tailed P < 0.0001 calculated by unpaired t-test). (C) Measurement of heart beats per minute in 72 hpf transgenic zebrafish larvae expressing GR DPR ubiquitously using the Hsp: Gal4 driver, subjected to heat shock at 48 hpf. Non-transgenic larvae subjected to heat shock were used as control. No significant difference was observed (N = 2, n = 5 larvae per condition, two-tailed P = 0.0679 by unpaired t-test). (D) Spontaneous coiling activity in 20 hpf embryos monitored over 20 min represented as percentage burst activity (percentage of total time during which embryos showed activity), which shows no difference in coiling activity between control and transgenic embryos since no DPRs would be expressed at this stage since Hb9 expression has just begun (N = 2, n = 40 larvae/genotype per condition).

Figure 5.

GR expression results in reduction of motor neuron length with increased cell death. (A) Example images of GFP-positive motor neurons in 48 hpf zebrafish larvae expressing GR DPR, and a non-transgenic larva as a control. Axons of motor neurons in GR100-expressing larvae appear shorter and less branched. (B) Measurement of the length of motor neurons in the spinal cord showed a significant decrease in motor neuron length when GR DPR is expressed (N = 2, measurement performed from seven embryos per condition, five ventral roots per embryo; two-tailed P < 0.0001 calculated by unpaired t-test). (C). Measurement of the length of motor neurons in the spinal cord before expression of the GR DPR at 48 hpf and at 80 hpf after 24 h of GR100 expression showed a significant decrease in motor neuron length only when GR DPR is expressed (N = 2, measurement performed from seven embryos per condition, five ventral roots per embryo; before heat shock group: two-tailed P = 0.7693 calculated by unpaired t-test; after heat shock group: two-tailed P < 0.0001 calculated by unpaired t-test). (D) Examples of GFP-positive motor neurons in 48 hpf and 80 hpf zebrafish larvae (prior to and after expressing GR DPR), with non-transgenic larvae served as a control. Axons of motor neurons abnormal after GR100 expression. (E) Examples of acridine orange–stained zebrafish larval spinal cords expressing GR DPR in neurons with larvae not expressing the DPR was used as a control. HuC expression was used to set limits while imaging, boundary of the cord is marked with black dotted lines. GR100 DPR expressing larvae show visibly more acridine orange positive cells (black dots, indicated by arrowheads), many of which located in the ventral region of the spinal cord. (F) Quantification of the number of acridine orange positive cells/larva/field in the spinal cord of larvae expressing GR DPR showed significantly greater number of AO positive cells in the spinal cord in comparison to larvae not expressing GR DPR (n = 12 larvae per genotype; two-tailed P = 0.0068 calculated by unpaired t-test).