Altered biometal homeostasis is associated with CLN6 mRNA loss in mouse neuronal ceroid lipofuscinosis

Abstract

Neuronal ceroid lipofuscinoses, the most common fatal childhood neurodegenerative illnesses, share many features with more prevalent neurodegenerative diseases. Neuronal ceroid lipofuscinoses are caused by mutations in CLN genes. CLN6 encodes a transmembrane endoplasmic reticulum protein with no known function. We characterized the behavioural phenotype of spontaneous mutant mice modeling CLN6 disease, and demonstrate progressive motor and visual decline and reduced lifespan in these mice, consistent with symptoms observed in neuronal ceroid lipofuscinosis patients. Alterations to biometal homeostasis are known to play a critical role in pathology in Alzheimer's, Parkinson's, Huntington's and motor neuron diseases. We have previously shown accumulation of the biometals, zinc, copper, manganese and cobalt, in CLN6 Merino and South Hampshire sheep at the age of symptom onset. Here we determine the physiological and disease-associated expression of CLN6, demonstrating regional CLN6 transcript loss, and concurrent accumulation of the same biometals in the CNS and the heart of presymptomatic CLN6 mice. Furthermore, increased expression of the ER/Golgi-localized cation transporter protein, Zip7, was detected in cerebellar Purkinje cells and whole brain fractions. Purkinje cells not only control motor function, an early symptomatic change in the CLN6 mice, but also display prominent neuropathological changes in mouse models and patients with different forms of neuronal ceroid lipofuscinoses. Whole brain fractionation analysis revealed biometal accumulation in fractions expressing markers for ER, Golgi, endosomes and lysosomes of CLN6 brains. These data are consistent with a link between CLN6 expression and biometal homeostasis in CLN6 disease, and provide further support for altered cation transporter regulation as a key factor in neurodegeneration.

Keywords: Biometal homeostasis; CLN6; Metal transporter; Neurodegeneration; Neuronal ceroid lipofuscinoses.

Fig. 1.. CLN6 mice demonstrate reduced lifespan and progressive motor and visual decline.

(A) Survival analysis of CLN6 (n = 24) and control C57BL/6 littermates (n = 22). Mice displaying full hind limb paralysis, over 15% weight loss from peak body weight or rotarod performance less than 10 seconds in 2 consecutive trials, were culled in accordance with ethical guidelines. P<0.0001 as calculated by the Log-rank (Mantel–Cox) Test. (B) CLN6 and control C57BL/6 mice were weighed twice-weekly over the duration of the study. P = 0.0029 for the effect of genotype on body weight as assessed by 2-way ANOVA. (C) Motor function was assessed by rotarod performance. P<0.0001 as assessed by 2-way ANOVA. Means became significantly different from the age of 34 weeks as assessed by post-hoc Bonferonni tests. (D) Visual function was determined by the visual placing test. Mice were graded on a scale from 0–2. P<0.0001 as assessed by 2-way ANOVA. Means became significantly different from the age of 42 weeks as assessed by post-hoc Bonferonni tests. (E) Grip strength was assessed weekly by the inverted grid test of grip strength. The longest time taken to fall from 2 consecutive trials each performed for 180 seconds was recorded. P = 0.0002 as assessed by 2-way ANOVA. Means became significantly different from the age of 43 weeks as assessed by post-hoc Bonferonni tests. For ease of visualization, all data (B–E) are expressed as mean values.

Fig. 2.. Spatial and temporal CLN6 transcript loss in CLN6 mice.

RNA isolated from CNS and peripheral tissues of 12 (A), 24 (B) and 32 (C) week-old control and CLN6 mice was reverse transcribed to cDNA and analysed for CLN6 transcript expression by qRT-PCR. Values are presented relative to tubulin expression. C, control; OB, olfactory bulb; CX, cortex; CB, cerebellum; BS, brainstem; SC, spinal cord; E, eye; L, liver; H, heart. P values determined by Student's t test. The box plots represent minimum, 25^th percentile, median, 75^th percentile and maximum relative expression values.

Fig. 3.. Biometals accumulate in the spinal cord, cortex, heart and liver of 12 week old CLN6 mice.

Metal concentrations in the CNS and peripheral tissues of 12 week old control and mutant CLN6 mice were measured using ICP-MS. The concentrations of zinc (A), manganese (B), cobalt (C) and copper (D) in each tissue are expressed as mean±S.D. values. C, control; OB, olfactory bulb; CX, cortex; CB, cerebellum; BS, brainstem; SC, spinal cord; E, eye; L, liver; H, heart. P values determined by Student's t test.

Fig. 4.. Biometals accumulate in the cerebellum and heart of 24 week old CLN6 mice.

Metal concentrations in the CNS and peripheral tissues of 24 week old control and mutant CLN6 mice were measured using ICP-MS. The concentrations of zinc (A), manganese (B), cobalt (C) and copper (D) in each tissue are expressed as mean±S.D. values. C, control; OB, olfactory bulb; CX, cortex; CB, cerebellum; BS, brainstem; SC, spinal cord; E, eye; L, liver; H, heart. P values determined by Student's t test.

Fig. 5.. Biometal changes in the cortex, spinal cord, olfactory bulb and heart in 32 week old CLN6 mice.

Metal concentrations in the CNS and peripheral tissues of 32 week old control and mutant CLN6 mice were measured using ICP-MS. The concentrations of zinc (A), manganese (B), cobalt (C) and copper (D) in each tissue are expressed as mean±S.D. values. C, control; OB, olfactory bulb; CX, cortex; CB, cerebellum; BS, brainstem; SC, spinal cord; E, eye; L, liver; H, heart. P values determined by Student's t test.

Fig. 6.. Zip7 transporter distribution is altered in the cerebellar Purkinje cell layer of pre-symptomatic CLN6 mice.

(A–G) Cerebellar homogenates (5–30 µg) isolated from 24-week old control or CLN6 mice (n = 3 per group) were immunoblotted with antibodies directed against members of the Zip (SLC39A) or ZnT (SLC30A) families of metal transporters. GAPDH, β-tubulin, total Akt or total ERK, as appropriate, were used as loading controls. Densitometry quantification was performed in ImageJ and metal transporter levels are expressed relative to those in control mice. (H) Representative immunoblots demonstrating metal transporter levels in cerebellum. (I) Immunofluorescent staining of coronal cerebellar sections (Bregma −5.8 mm) from 24 week-old control and CLN6 mice was used to assess localized variations in Zip7, Zip8 and ZnT7 expression. Arrows indicate Purkinje cells. Nuclei were stained blue with DAPI. Images are representative of 3 mice per genotype. Scale bars: 100 µm.

Fig. 7.. Biometals accumulate in lysosomes and ER in presymptomatic CLN6 mouse brain.

Sucrose density gradient fractions from 24 week old whole mouse brain were analysed for metal content by ICP-MS. Data are expressed as the mean±S.D of the ratio of zinc (A), manganese (B), cobalt (C) and copper (D) concentrations in CLN6 brains relative to control brains from 3 independent experiments. Dotted lines represent equal metal content in control and CLN6 brains. Organelles corresponding to fraction numbers are based on western blotting analyses. L, lysosomes; EE, early endosomes; ER, endoplasmic reticulum; G, Golgi apparatus; N, nucleus. (E) Representative immunoblot of Zip7 protein present in control and CLN6 fractions.