A novel porcine model of CLN3 Batten disease recapitulates clinical phenotypes

Abstract

Mouse models of CLN3 Batten disease, a rare lysosomal storage disorder with no cure, have improved our understanding of CLN3 biology and therapeutics through their ease of use and a consistent display of cellular pathology. However, the translatability of murine models is limited by disparities in anatomy, body size, life span and inconsistent subtle behavior deficits that can be difficult to detect in CLN3 mutant mouse models, thereby limiting their use in preclinical studies. Here, we present a longitudinal characterization of a novel miniswine model of CLN3 disease that recapitulates the most common human pathogenic variant, an exon 7-8 deletion (CLN3Δex7/8). Progressive pathology and neuron loss is observed in various regions of the CLN3Δex7/8 miniswine brain and retina. Additionally, mutant miniswine present with retinal degeneration and motor abnormalities, similar to deficits seen in humans diagnosed with the disease. Taken together, the CLN3Δex7/8 miniswine model shows consistent and progressive Batten disease pathology, and behavioral impairment mirroring clinical presentation, demonstrating its value in studying the role of CLN3 and safety/efficacy of novel disease-modifying therapeutics.

Keywords: Animal disease models; JNCL; Neuronal ceroid lipofuscinosis; Neuropediatric disease.

Fig. 1.

Study design and gait score. (A) Study design showing time points for behavior testing and histopathology throughout the study period. (B) Plotted is the PCA gait score for front feet obtained from combined-sex datasets. CLN3^Δex7/8 animals have a significantly different gait than wild-type animals at all time points. (C) Description of variables contributing to gait scores for combined-sex datasets. (D) Plotted is the foot area for combined-sex datasets. CLN3^Δex7/8 miniswine have a significantly decreased footfall size compared to that of wild-type swine at all time points. (E,F) Stance center-of-pressure (COP) variables for combined-sex datasets. CLN3^Δex7/8 miniswine have less variability in stance COP trajectories, indicative of a cautious balancing strategy and more-controlled stance. Mean±s.e.m. Two-way ANOVA with uncorrected Fisher's LSD post-hoc test. *P≤0.05, **P≤0.01, ***P≤0.001. B, both, R; right, L; left; SD, standard deviation; CV, coefficient of variation. Numbers of animals tested are listed in Table S4.

Fig. 2.

CLN3^Δex7/8 animals show photopic and scotopic visual deficits by 30 months of age as measured by electroretinography (ERG). (A-D) Photopic data. At 30 months of age, CLN3^Δex7/8 miniswine begin to show a progressive decline in photopic a-wave (A) and b-wave (B) amplitudes. Latency delays arise at 30 months for both a-waves (C) and b-waves (D). (E-H) Scotopic data. Scotopic a-wave (E) and b-wave (F) amplitudes in CLN3^Δex7/8 miniswine begin to decline at 42 months of age and are extinguished at 48 months of age. Latency delays arise at 30 months for both a-wave (G) and b-wave (H) in all CLN3^Δex7/8 miniswine. At 42 and 48 months, WT n=two males; CLN3^Δex7/8 n=3 (two males, one female). Mean±s.e.m. Two-way ANOVA, Fisher's LSD post-hoc test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Photopic: 8.0 cd s/m² flash at 2.0 Hz (cone predominant). Scotopic: 8.0 cd s/m² flash at 0.1 Hz (bright flash standard combined response-mixed rods and cones). Absolute values for a-wave amplitudes are shown. Numbers of animals tested are listed in Table S4.

Fig. 3.

In CLN3^Δex7/8 miniswine a- and b-waves reduce with age. Mixed rod and cones responses are extinguished by 42 months (right); cone predominant responses are extinguished by 48 months. The decline in a- and b- amplitudes is readily seen in a- and b-waves of CLN3^Δex7/8 miniswine aged between 30 and 48 months. At 48 months, the cone predominant waveforms are completely extinguished, only background noise is recorded. At 42 months, the mixed rod and cone responses are nonrecordable. Photopic: 8.0 cd s/m² flash at 2.0 Hz (cone predominant). Scotopic: 8.0 cd s/m² flash at 0.1 Hz (bright flash standard combined response-mixed rods and cones). All wave recordings were obtained from the same CLN3^Δex7/8 male.

Fig. 4.

CLN3^Δex7/8 animals show retinal thinning as evidenced by loss of cells at 48 months of age. (A) Comparison of retinal images from WT and CLN3^Δex7/8 miniswine aged 48 months. Loss of photoreceptors (PR) is seen in 48-month-old CLN3^Δex7/8 miniswine (right) compared with WT swine (left). Measured were retinas of 36 and 48-month-old miniswine. (B,C) Plotted is the percentage of the width of the outer nuclear layer (ONL) (A) and the ONL cell body count, i.e. loss of photoreceptors, (C) for WT and CLN3^Δex7/8 miniswine aged 36 and 48 months. At 36 months: n=5 (two females, three males); at 48 months: n=3 (one female, two males). Mean±s.e.m. Nested t-tests. *P≤0.05. GCL, ganglion cell layer; INL, inner nuclear layer.

Fig. 5.

CLN3^Δex7/8 animals show accumulation of mitochondrial ATP synthase subunit C (SubC) in several brain regions. (A) Nissl-stained coronal section of a miniswine brain indicating the anatomical location of somatosensory cortex (SSC), motor cortex (MC), ventral posteromedial and ventral posterolateral nuclei (VPM-VPL) of the thalamus, and CA2-CA3 of hippocampus. (B-I) Subunit C accumulation was evident in CLN3^Δex7/8 miniswine, beginning at 2 months of age in the somatosensory cortex (B,C), motor cortex (D,E), CA2-CA3 (F,G) and the VPM/VPL nuclei (H,I). Accumulation was persistent at all time points measured. Mean±s.e.m., unpaired t-test, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Scale bar: 200 µm. Numbers of animals tested are listed in Table S4.

Fig. 6.

CLN3^Δex7/8 miniswine show microglia and astrocyte reactivity in several brain regions. (A-F) Increased IBA1-positive (IBA1+) microglia soma size was evident at 14 to 36 months of age in the somatosensory cortex (A,B) and motor cortex (C,D) of CLN3^Δex7/8 animals. Microglial reactivity was not evident in ventral posteromedial and ventral posterolateral nuclei (VPM-VPL) (E,F). (G,H) GFAP-positive astrocytosis was evident between 6 and 36 months of age in the VPM/VPL of the thalamus of CLN3^Δex7/8 animals. Mean±s.e.m., nested t-test (IBA1) or unpaired t-test (GFAP), *P≤0.05, **P≤0.01, ****P≤0.0001. Scale bars: 200 µm. Numbers of animals tested are listed in Table S4.