Evaluation of the Potential Role of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Niemann-Pick Disease, Type C1

Abstract

Niemann-Pick disease, type C1, is a cholesterol storage disease where unesterified cholesterol accumulates intracellularly. In the cerebellum this causes neurodegeneration of the Purkinje neurons that die in an anterior-to-posterior and time-dependent manner. This results in cerebellar ataxia as one of the major outcomes of the disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a significant role in the regulation of serum cholesterol levels by modulating LDL receptor levels on peripheral tissues. In the central nervous system, PCSK9 may have a similar effect on the closely related VLDL and ApoE2 receptors to regulate brain cholesterol. In addition, regulation of VLDLR and ApoER2 by PCSK9 may contribute to neuronal apoptotic pathways through Reelin, the primary ligand of VLDLR and ApoER2. Defects in reelin signaling results in cerebellar dysfunction leading to ataxia as seen in the Reeler mouse. Our recent findings that Pcsk9 is expressed ~8-fold higher in the anterior lobules of the cerebellum compared to the posterior lobule X, which is resistant to neurodegeneration, prompted us to ask whether PCSK9 could play a role in NPC1 disease progression. We addressed this question genetically, by characterizing NPC1 disease in the presence or absence of PCSK9. Analysis of double mutant Pcsk9^-/-/Npc1^-/- mice by disease severity scoring, motor assessments, lifespan, and cerebellar Purkinje cell staining, showed no obvious difference in NPC1 disease progression with that of Npc1^-/- mice. This suggests that PCSK9 does not play an apparent role in NPC1 disease progression.

Keywords: ApoER2; NPC1 KO mouse; Niemann–Pick C; PCSK9; VLDLR; lysosomal storage; neurodegeneration.

Figure 1

Weight and disease progression in proprotein convertase subtilisin/kexin type 9 deficient mice (Pcsk9^-/-) in the context of NPC1 disease. Male (A) and female (B) mice were weighed weekly. Pcsk9^-/-/Npc1^ctrl mice had a normal weight gain profile. Npc1 mutant mice begin to lose weight at ~6 weeks of age and continued thereafter. There was no significant difference observed between Pcsk9^ctrl/Npc1^-/- and Pcsk9^-/-/Npc1^-/- mice. Male (C) and female (D) mice were assessed on a weekly basis for disease severity: grooming, gait, extent of kyphosis, hindlimb clasp and ability to walk along the ledge of the cage. As expected, Pcsk9^-/-/Npc1^ctrl mice displayed negligible scores throughout the course of the assessments. The Npc1 mutant mice exhibited an increase in disease severity starting at ~6 weeks of age and continued thereafter. No significant difference was observed between Pcsk9^ctrl/Npc1^-/- and Pcsk9^-/-/Npc1^-/- groups.

Figure 2

Motor coordination in Pcsk9^-/- mice in the context of NPC1 disease. Using the AccuScan activity box, the horizontal activity (A), walking speed (B), and vertical activity (C) of the mice were recorded on a weekly basis. Results from 5, 7 and 9 weeks are plotted here. As expected, Pcsk9^-/-/Npc1^ctrl showed consistent measurements of all activity across all timepoints. In contrast, Pcsk9^ctrl/Npc1^-/- and Pcsk9^-/-/Npc1^-/- groups displayed decreased horizontal activity and walking speed starting at week 7, which was worse and more significant at 9 weeks. The average number of vertical movements of both Pcsk9^ctrl/Npc1^-/- and Pcsk9^-/-/Npc1^-/- groups was significantly decreased by week 9. Asterix indicate significance levels, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Mouse survival rates in Pcsk9^-/- mice in the context of NPC1 disease. Male and female mutant mice were assessed for survival. The end of life timepoint was assessed based on severity of motor impairment. No significant difference in survival was observed between male (A) or female (B) Pcsk9^ctrl/Npc1^-/- and Pcsk9^-/-/Npc1^-/- mice.

Figure 4

Purkinje cell loss in Pcsk9^-/- mice in the context of NPC1 disease. Cerebellar sections from 7-week old Pcsk9^-/-/Npc1^+/+ (A), Pcsk9^-/-/Npc1^-/- (B) and Pcsk9^+/+/Npc1^-/- (C) mice were analyzed by indirect immunohistochemical staining. Purkinje neurons were stained for calbindin (red) and counterstained with DAPI (blue) to distinguish the granule cell layer from the molecular layer. In Pcsk9^-/-/Npc1^+/+ sections (A), Purkinje neurons were found generally in all lobules and especially in lobule I/II. In Pcsk9^-/-/Npc1^-/- (B) and Pcsk9^+/+/Npc1^-/- (C) sections, a marked decrease of calbindin staining in the anterior lobules (I-IV/V) indicated Purkinje neuron death consistent with the level of NPC1 disease at this age. No obvious difference was observed in the calbindin staining pattern between Pcsk9^-/-/Npc1^-/- and Pcsk9^+/+/Npc1^-/- mice, suggesting that deletion of Pcsk9 does not appear to alter Purkinje cell death progression in the context of NPC1 disease. Bar = 200 μm. Arrows point to Purkinje neuron cell bodies in panel A, and their absence in panels B, C.

Figure 5

Serum liver enzyme levels in Pcsk9^-/- mice in the context of Niemann–Pick disease, type C1 (NPC1) disease. In order to assess liver function, serum was collected from control and mutant mice at 7–8 weeks of age. All three enzymes, (A) alkaline phosphatase (AP), (B) alanine aminotransferase (ALT) and (C) aspartate aminotransferase (AST), were elevated in the Npc1^-/- mice compared to control mice. The levels of AP and AST between Pcsk9^-/-/Npc1^-/- and Pcsk9^+/+/Npc1^-/- were not statistically different, whereas the ALT was slightly reduced in the Pcsk9^-/-/Npc1^-/- mice. These results demonstrate that liver function was affected similarly in Npc1^-/- mice in the presence or absence of PCSK9.