Circadian profiling in two mouse models of lysosomal storage disorders; Niemann Pick type-C and Sandhoff disease

Abstract

Sleep and circadian rhythm disruption is frequently associated with neurodegenerative disease, yet it is unclear how the specific pathology in these disorders leads to abnormal rest/activity profiles. To investigate whether the pathological features of lysosomal storage disorders (LSDs) influence the core molecular clock or the circadian behavioural abnormalities reported in some patients, we examined mouse models of Niemann-Pick Type-C (Npc1 mutant, Npc1(nih)) and Sandhoff (Hexb knockout, Hexb(-/-)) disease using wheel-running activity measurement, neuropathology and clock gene expression analysis. Both mutants exhibited regular, entrained rest/activity patterns under light:dark (LD) conditions despite the onset of their respective neurodegenerative phenotypes. A slightly shortened free-running period and changes in Per1 gene expression were observed in Hexb(-/-) mice under constant dark conditions (DD); however, no overt neuropathology was detected in the suprachiasmatic nucleus (SCN). Conversely, despite extensive cholesterol accumulation in the SCN of Npc1(nih) mutants, no circadian disruption was observed under constant conditions. Our results indicate the accumulation of specific metabolites in LSDs may differentially contribute to circadian deregulation at the molecular and behavioural level.

Keywords: Ataxia; Circadian; Lysosome storage disorder; Mouse mutant.

Fig. 1

Wheel-running behaviour in Npc1^nih mutants in LD and DD. (A) Npc1^nih activity under a 12:12 LD cycle (from 7 weeks of age) followed by DD (Npc1^nih screen 1; Fig. S1 for details) versus WT controls. Each horizontal line represents a double-plot of the activity profile recorded over 24 h. Activity bars are displayed in 10 min bins and yellow shading indicates light exposure. (B) Npc1^nih mutants show significantly reduced activity over each day in LD (n = 11 each genotype, Npc1^nih screens 1 and 2 combined) and DD (n = 5, Npc1^nih screen 1) compared to WT controls. (C) Free-running activity onsets do not differ between Npc1^nih mutants and WT controls (n = 5, Npc1^nih screen 1). Data presented as mean ± SEM; *p < 0.01, ***p < 0.0001, ANOVA.

Fig. 2

Wheel-running behaviour in Hexb^−/− mutants in LD and DD. (A) Hexb^−/− activity under a 12:12 LD cycle (from 7 weeks of age) followed by DD (Hexb^−/− screen; Fig. S1 for details) versus WT controls. (B) Hexb^−/− mutants show no difference in activity over each day in LD and at only one hour during DD (n = 6, Hexb screen 1) compared to WT controls (n = 7). (C) Free-running activity onsets under DD are significantly earlier in Hexb^−/− mutants compared to WT. Data presented as mean ± SEM; *p < 0.05, ANOVA

Fig. 3

Wheel-running of Hexb^−/− mutants under constant light (LL). After DD, Hexb^−/− mutants and controls were place in LL (Fig. S1; Hexb^−/− screen). (A and B) One third (2 of 6) of the Hexb^−/− mutants displayed period lengthening as expeced in WT mice. (C) Two-thirds (4 out of 6) Hexb^−/− mutants show a photoperiod of less than 24 h under LL.

Fig. 4

Phase-resetting behaviour in Npc1^nih mutants. (A) Npc1^nih activity onset following a six-hour phase advance was similar to WT for the first 2 days, however increasing onset variability was observed, indicating rhythm instability (n = 5). (B) Average activity onsets in Npc1^nih mice were significantly different from WT 3 days after the phase advance (indicated with an arrow). Data presented as mean ± SEM; *p < 0.01, ***p < 0.0001, ANOVA.

Fig. 5

Passive-infrared (PIR) tracking in Npc1^nih mutants in a 12:12 LD cycle. Each row represents a double-plot of the activity profile recorded over 24 h. (A) Activity bars are displayed in 10 min bins and yellow shading indicates light exposure. (B) Average activity over day 3 of the PIR screen. Npc1^nih mutants show significant increase in light-phase activity compared to WT. **p < 0.01, ***p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Neuropathology of LSD mouse models. (A–D) Representative light microscopy images of Periodic Acid–Schiff (PAS)-stained brain sections from Hexb^−/− mutants and WT controls at 12 weeks of age. Stained sections are taken from the dentate gyrus (A, B) and the SCN (C, D). The glycosphingolipid (GSL) storage products stained with PAS appear pink. Arrows indicate PAS-positive neuronal inclusions in Hexb^−/− mutants. (E, F) Representative fluorescent microscopy images of filipin-stained SCN sections from Npc1^nih mutant and WT controls at 11 weeks of age. Scale bars: 100 μm.

Fig. 7

Clock gene expression in Hexb^−/− mutants by RT-qPCR. (A) No significant difference in selected clock gene expression was observed in the SCN of Hexb^−/− mutants versus WT at CT6 under DD (n = 4–5). Hexb^−/− mutants show a significant differences in the expression of Per1 in the liver at ZT6 under a 12:12 LD cycle (B) and at CT6 under DD compared (C) to WT (n = 4–5). Data are presented as mean ± SEM; *p = <0.001, ANOVA.

Fig. 8

Retinal histopathology in Hexb^−/− mutants. (A) Representative immunofluorescence images of Brn3a and melanopsin expression in WT ventral retina (n = 3). (B) Representative immunofluorescence images of Brn3a and melanopsin expression in Hexb^−/− ventral retina (n = 3). (C) Some dorsal areas of Hexb^−/− retina showed more significant disruption of Brn3a labelling. Scale bars: 50 μm.