GDAP2 mutations implicate susceptibility to cellular stress in a new form of cerebellar ataxia

Abstract

Autosomal recessive cerebellar ataxias are a group of rare disorders that share progressive degeneration of the cerebellum and associated tracts as the main hallmark. Here, we report two unrelated patients with a new subtype of autosomal recessive cerebellar ataxia caused by biallelic, gene-disruptive mutations in GDAP2, a gene previously not implicated in disease. Both patients had onset of ataxia in the fourth decade. Other features included progressive spasticity and dementia. Neuropathological examination showed degenerative changes in the cerebellum, olive inferior, thalamus, substantia nigra, and pyramidal tracts, as well as tau pathology in the hippocampus and amygdala. To provide further evidence for a causative role of GDAP2 mutations in autosomal recessive cerebellar ataxia pathophysiology, its orthologous gene was investigated in the fruit fly Drosophila melanogaster. Ubiquitous knockdown of Drosophila Gdap2 resulted in shortened lifespan and motor behaviour anomalies such as righting defects, reduced and uncoordinated walking behaviour, and compromised flight. Gdap2 expression levels responded to stress treatments in control flies, and Gdap2 knockdown flies showed increased sensitivity to deleterious effects of stressors such as reactive oxygen species and nutrient deprivation. Thus, Gdap2 knockdown in Drosophila and GDAP2 loss-of-function mutations in humans lead to locomotor phenotypes, which may be mediated by altered responses to cellular stress.

Figure 1

Main neuropathological changes within the brain of Patient AW. (A) Severe cerebellar atrophy (arrows) showing that a large part of the occipital lobes (OL) is not covered by the cerebellum as usual. (B) Control showing a normal cerebellum (arrows) covering the occipital lobes. (C) Cerebellar foliae showing the disappearance of the Purkinje cells (the arrows point to the layer where Purkinje cells should be). Thinning of the inner granular layer (IGL). (D) Control case showing the layer of the Purkinje cells (arrows) and the inner granular layer. (E) Dorsal part of the main olivary nucleus in the medulla oblongata (arrow). A comparison with a control case (F) highlights a slight loss and an atrophy of the neurons. (F) Control case showing larger and more numerous neurons in the same area. (G) Neuronal losses in the thalamic pulvinar. (H) Normal amounts of neurons (arrows) in a control pulvinar. (I) Neuronal loss and atrophy in the zona compacta of the substantia nigra. (J) Control case. The arrow points towards one of the many melanin-containing neurons. (C–J) Paraffin sections, cresyl violet staining. Scale bars = 50 µm.

Figure 2

Modelling loss of GDAP2 function inDrosophila decreases survival. (A) High conservation of protein organization and amino acid sequence between human GDAP2 andDrosophila Gdap2. Conserved protein domains include the macro (red, 73% similarity) and CRAL-TRIO (blue, 68% similarity) domain. (B) RelativeGdap2 expression in male Control-1 (dark grey, genotype:UAS-Dicer-2;Actin-Gal4/+), Gdap2 RNA-1 (dark blue, genotype:UAS-Dicer-2;Actin-Gal4/+;Gdap2 RNAi-1/+), Control-2 (light grey, genotype:UAS-Dicer-2;Actin-Gal4/+) and Gdap2 RNAi-2 (light blue, genotype:UAS-Dicer-2;Actin-Gal4/Gdap2 RNAi-2) flies determined by qPCR. Min-to-max boxplots represent three independent biological replicates. **P <0.01, ***P <0.001. (C andD) Colour code as inB and in legend on the right. (C) Eclosion rate (in %) of Control-1, Gdap2 RNA-1, Control-2 and Gdap2 RNAi-2 male flies. Min-to-max boxplots represent three independent biological replicates. ***P <0.001. (D) Kaplan Meijer curve showing survival (in %) over days past eclosion of male Control-1, Gdap2 RNA-1, Control-2 and Gdap2 RNAi-2 flies. Error bars indicate standard error (SE).Gdap2 knockdown dramatically diminishes survival. The log-rank (Mantel-Cox) test confirmed that survival significantly differs (Control-1 versus Gdap2 RNAi-1:P <0.001 and Control-2 versus Gdap2 RNAi-2:P <0.001).

Figure 3

Drosophila GDAP2 knockdown models show aberrant locomotor behaviour. (A)Drosophila escape response, assessed in the island assay. Graphs show per cent of flies that remain on the platform over time (10 s). Two-day-old male Control-1 (dark grey), Gdap2 RNA-1 (dark blue), Control-2 (light grey) and Gdap2 RNAi-2 (light blue) flies were tested. Plotted is average and SEM of three independent biological replicates. (B) Floating bars depicting minimum, maximum and mean area under curve based on the graphs shown inA, **P <0.01, ***P <0.001. (C) Locomotion trajectories of representative flies of the same genotypes as inA andB. Two-day-old male flies were recorded for 7 min at 10 frames per second in a circular arena. (D) Total distance (in cm) of walk in the 7 min of locomotion tracking. (E) Average speed (in cm/s) of the indicated genotypes during the 7 min of locomotion tracking. Genotypes are specified in theFig. 2 legend and in the ‘Materials and methods’ section. Min-to-max boxplots represent data of three independent biological replicates, ***P <0.001.

Figure 4

Drosophila GDAP2 knockdown models are susceptible to cellular stress. (A–D) Relative Gdap2 expression levels are reduced upon starvation (A andB) and 25 mM paraquat exposure (C andD) in two control strains. Plotted are min-to-max boxplots of 4–6 biological replicates. *P <0.05, **P <0.01, ***P <0.001. (E–H) Kaplan Meijer curve showing survival (in %) over time upon exposure to the indicated stressors. (E andF) Starvation (starved), (G andH) oxidative stress induced by 25 mM paraquat (25 mM PQ). Male flies were tested, genetic conditions, treated (light blue or light grey lines) and untreated (dark blue or dark grey lines), are specified in the legend on theright. Gdap2 knockdown flies (Gdap2 RNAi-1 and RNAi-2) lived significantly shorter upon nutrient starvation (E andF) or 25 mM paraquat exposure (G andH) compared to treated controls and untreated Gdap2 knockdown and control flies.P-values are indicated above each panel, genotypes are specified in theFig. 2 legend and in the ‘Materials and methods’ section. Error bars represent SE. Log-rank (Mantel-Cox) test was used to determine whether survival curves significantly differ from each other.