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

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.


Introduction
Niemann-Pick disease, type C (NPC) is an autosomal recessive, lysosomal storage disorder that affects~1:120,000 births [1,2]. Patients with NPC initially present with hepatosplenomegaly that is followed by progressive neurodegeneration leading to cerebellar ataxia and dementia [1,3] with patients typically succumbing to the disease in the second decade of life [4]. NPC is caused by mutations in NPC1 or NPC2, which encode for proteins that are involved in the transport of regulation of ApoER2 levels in the brain [37]. VLDLR and ApoER2 are implicated in neuronal processes including cerebellar development and synaptic plasticity through Reelin signaling [38]. In humans, loss of VLDLR results in a non-progressive cerebellar ataxia phenotype [39] and mutations in RELN cause cerebellar hypoplasia [40] similar to that of the Reeler mouse [41]. In addition, canonical and non-canonical signaling pathways of Reelin may contribute to its neuroprotective function, for example in the regulation of tau phosphorylation in the context of Alzheimer disease pathology [42,43]. Hence, regulation of VLDLR and ApoER2 by PCSK9 may contribute to Reelin signaling and increased neuroprotection.
Given our recent results showing higher expression of Pcsk9 in the anterior lobules of the cerebellum where degeneration of the Purkinje neurons begins in NPC disease and the possible association with neuronal modulators ApoER2 and VLDLR, we hypothesized that decreased expression of Pcsk9 would increase surface expression of ApoER2 and VLDLR and thus confer neuroprotection of the Purkinje neurons in the cerebellum. To test this, we characterized the Npc1 -/mouse phenotype in the presence and absence of the Pcsk9 gene.

Knockout of Pcsk9 Did not Significantly Alter Weight Progression in Affected Npc1 -/-Mice
Mouse weights were recorded on a weekly basis from 4-11 weeks of age. Control male (n = 13) and female mice (n = 15) gained weight as expected for control male and female mice, respectively ( Figure 1A,B). No obvious differences were seen between the Pcsk9 +/+ and Pcsk9 +/mice and these data were combined as Pcsk9 ctrl . Single knockout Npc1 -/-/Pcsk9 ctrl male (n = 10) and female (n = 14) mice had an initial normal, but slightly reduced, weight-gain followed by weight loss starting at about 6-7 weeks of age as expected for the Npc1 -/phenotype [44,45]. In the double knockout Npc1 -/-/Pcsk9 -/mice, both male (n = 6) and female (n = 7) mice were similar to the Npc1 -/-/Pcsk9 ctrl mice, showing a similar weight loss pattern consistent with the phenotype of Npc1 -/mutant mice alone ( Figure 1A,B). and female mice (n = 15) gained weight as expected for control male and female mice, respectively ( Figure 1A,B). No obvious differences were seen between the Pcsk9 +/+ and Pcsk9 +/mice and these data were combined as Pcsk9 ctrl . Single knockout Npc1 -/-/Pcsk9 ctrl male (n = 10) and female (n = 14) mice had an initial normal, but slightly reduced, weight-gain followed by weight loss starting at about 6-7 weeks of age as expected for the Npc1 -/phenotype [44,45]. In the double knockout Npc1 -/-/Pcsk9 -/mice, both male (n = 6) and female (n = 7) mice were similar to the Npc1 -/-/Pcsk9 ctrl mice, showing a similar weight loss pattern consistent with the phenotype of Npc1 -/mutant mice alone ( Figure 1A,B). 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. 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.

NPC1 Neurological Disease Progression Was Not Affected by Disruption of Pcsk9
In order to evaluate the potential roles of Pcsk9 in Npc1 -/mice, control, single mutant Npc1 -/-/Pcsk9 ctrl and double mutant Npc1 -/-/Pcsk9 -/mice were assessed for disease progression on a weekly basis from 4-11 weeks of age using a composite disease severity scale [46]. No obvious differences were seen between the Pcsk9 +/+ and Pcsk9 +/mice and the data were combined as Pcsk9 ctrl . The composite disease severity score did not show a significant difference between single mutant Npc1 -/-/Pcsk9 ctrl mice and double mutant Npc1 -/-/Pcsk9 -/mice ( Figure 1C,D). The lack of significant difference between single and double mutants was observed in both male and female cohorts ( Figure 1C,D). Control mice (Npc1 ctrl /Pcsk9 -/-) exhibited only baseline scores that remained constant throughout the observation timeframe.
Assessment of mouse activity and motor coordination was performed in an AccuScan Activity box. Horizontal activity, walking speed and vertical activity were recorded. Rearing ability, as quantified by number of vertical movements, was used as an indication of motor coordination to complement the ledge test of the composite disease severity score. Npc1 -/mice have a well-characterized motor impairment which did not appear to be significantly altered in the absence of Pcsk9 (Figure 2). Both Npc1 -/-/Pcsk9 ctrl and Npc1 -/-/Pcsk9 -/cohorts had similar lower average number of vertical movements than control mice at week 9, consistent with the disease severity. This trend was consistent in both male and female mice. The other measurements of movement (horizontal activity and speed) were likewise equally affected in both Npc1 -/-/Pcsk9 ctrl and Npc1 -/-/Pcsk9 -/groups. quantified by number of vertical movements, was used as an indication of motor coordination to complement the ledge test of the composite disease severity score. Npc1 -/mice have a wellcharacterized motor impairment which did not appear to be significantly altered in the absence of Pcsk9 ( Figure 2). Both Npc1 -/-/Pcsk9 ctrl and Npc1 -/-/Pcsk9 -/cohorts had similar lower average number of vertical movements than control mice at week 9, consistent with the disease severity. This trend was consistent in both male and female mice. The other measurements of movement (horizontal activity and speed) were likewise equally affected in both Npc1 -/-/Pcsk9 ctrl and Npc1 -/-/Pcsk9 -/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 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.

Cerebellar Analysis of Purkinje Neurons by Immunohistochemical Staining
To exclude the possibility that increased cholesterol uptake in the liver, as expected in Pcsk9 -/mice [32], could lead to a more severe liver disease in Npc1 -/-, that would mask a possible neurological benefit, we evaluated Purkinje neuron survival and liver disease in Npc1 -/-/Pcsk9 -/and Npc1 -/-/Pcsk9 ctrl mice. Immunostaining of calbindin, as a specific marker for Purkinje neurons, in cerebellar sections from 7-week old Npc1 +/+ /Pcsk9 -/mice showed an intact distribution of Purkinje neurons throughout the lobules of the cerebellum ( Figure 4A). This pattern is consistent with a healthy cerebellar phenotype with respect to Purkinje neurons [16] and demonstrates that lack of Pcsk9 did not appear to affect these neurons in Npc1 +/+ mice although subtle changes may have occurred that were not observed. In contrast, staining from 7-week old Npc1 -/-/Pcsk9 -/mice showed a significant reduction of Purkinje neurons in the anterior lobules of the cerebellum ( Figure 4B), consistent with the expected Purkinje neuron loss at this age during disease progression of NPC [16]. As a control, similar staining of a 7-week old Npc1 -/-/Pcsk9 +/+ mouse was evaluated and showed that Purkinje neuron loss appeared similar in Npc1 -/mice irrespective of the Pcsk9 genotype ( Figure 4C).

Cerebellar Analysis of Purkinje Neurons by Immunohistochemical Staining
To exclude the possibility that increased cholesterol uptake in the liver, as expected in Pcsk9 -/mice [32], could lead to a more severe liver disease in Npc1 -/-, that would mask a possible neurological benefit, we evaluated Purkinje neuron survival and liver disease in Npc1 -/-/Pcsk9 -/and Npc1 -/-/Pcsk9 ctrl mice. Immunostaining of calbindin, as a specific marker for Purkinje neurons, in cerebellar sections from 7-week old Npc1 +/+ /Pcsk9 -/mice showed an intact distribution of Purkinje neurons throughout the lobules of the cerebellum ( Figure 4A). This pattern is consistent with a healthy cerebellar phenotype with respect to Purkinje neurons [16] and demonstrates that lack of Pcsk9 did not appear to affect these neurons in Npc1 +/+ mice although subtle changes may have occurred that were not observed. In contrast, staining from 7-week old Npc1 -/-/Pcsk9 -/mice showed a significant reduction of Purkinje neurons in the anterior lobules of the cerebellum ( Figure 4B), consistent with the expected Purkinje neuron loss at this age during disease progression of NPC [16]. As a control, similar staining of a 7-week old Npc1 -/-/Pcsk9 +/+ mouse was evaluated and showed that Purkinje neuron loss appeared similar in Npc1 -/mice irrespective of the Pcsk9 genotype ( Figure 4C).

Discussion
The role of PCSK9 in serum cholesterol regulation has been well established. Indeed, since its discovery in 2003, our current knowledge represents the complement of comprehensive basic and translational science, leading ultimately to an effective treatment of hypercholesterolemia [30]. However, until more comprehensive studies on PCSK9 are undertaken to explore its role in the central nervous system (CNS), treatment of PCSK9-regulating drugs may affect the CNS in unseen and unwanted ways.
The exact role of PCSK9 in the CNS remains unknown. However, due to its well characterized cell biological mechanism in regulating the levels of the LDL receptor in the periphery, it is expected to function similarly in the brain. Studies have shown it can bind and regulate the degradation of the VLDL and ApoE2 receptors in a similar manner to that of the LDL receptor. In addition, since mature neurons receive their cholesterol from astrocytes through VLDLR and ApoER2, it is predicted that PCSK9 could contribute to brain cholesterol metabolism by regulating these receptors. In the adult mouse brain, it has been reported that PCSK9 is not involved in LDLR degradation in the cortex and hippocampus [36]. However, Pcsk9 is increased in the dentate gyrus after transient cerebral ischemia and in a mouse model of hyperlipidemia where neuronal apoptosis occurs in the hippocampus [47,48]. In both cases, reduced LDLR levels and increased neuronal apoptosis in these tissues appear to correlate with the increase in Pcsk9. This suggests that in the context of disease or injury, levels of PCSK9 may regulate neuronal apoptotic signaling pathways. Based on these observations we therefore sought to test whether PCSK9 plays a role in NPC1, a disease of cholesterol metabolism.
Our studies however have shown that PCSK9 does not appear to play a role in NPC1 pathology even though we found expression differences in the lobules susceptible and resistant to Purkinje neuron death [16]. In all parameters measured (disease severity score, weight, lifespan, selected motor functions and Purkinje neuron staining and serum liver enzymes), we did not discern an obvious difference in NPC1 disease progression in mice with or without PCSK9. Perhaps the levels of expression of PCSK9 are too low in the adult cerebellum to affect receptor levels especially in the Purkinje neurons layer, or perhaps specificity and sensitivity of binding to VLDLR or ApoER2 are different in vivo in the context of a cholesterol trafficking defect. 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.

Discussion
The role of PCSK9 in serum cholesterol regulation has been well established. Indeed, since its discovery in 2003, our current knowledge represents the complement of comprehensive basic and translational science, leading ultimately to an effective treatment of hypercholesterolemia [30]. However, until more comprehensive studies on PCSK9 are undertaken to explore its role in the central nervous system (CNS), treatment of PCSK9-regulating drugs may affect the CNS in unseen and unwanted ways.
The exact role of PCSK9 in the CNS remains unknown. However, due to its well characterized cell biological mechanism in regulating the levels of the LDL receptor in the periphery, it is expected to function similarly in the brain. Studies have shown it can bind and regulate the degradation of the VLDL and ApoE2 receptors in a similar manner to that of the LDL receptor. In addition, since mature neurons receive their cholesterol from astrocytes through VLDLR and ApoER2, it is predicted that PCSK9 could contribute to brain cholesterol metabolism by regulating these receptors. In the adult mouse brain, it has been reported that PCSK9 is not involved in LDLR degradation in the cortex and hippocampus [36]. However, Pcsk9 is increased in the dentate gyrus after transient cerebral ischemia and in a mouse model of hyperlipidemia where neuronal apoptosis occurs in the hippocampus [47,48]. In both cases, reduced LDLR levels and increased neuronal apoptosis in these tissues appear to correlate with the increase in Pcsk9. This suggests that in the context of disease or injury, levels of PCSK9 may regulate neuronal apoptotic signaling pathways. Based on these observations we therefore sought to test whether PCSK9 plays a role in NPC1, a disease of cholesterol metabolism.
Our studies however have shown that PCSK9 does not appear to play a role in NPC1 pathology even though we found expression differences in the lobules susceptible and resistant to Purkinje neuron death [16]. In all parameters measured (disease severity score, weight, lifespan, selected motor functions and Purkinje neuron staining and serum liver enzymes), we did not discern an obvious difference in NPC1 disease progression in mice with or without PCSK9. Perhaps the levels of expression of PCSK9 are too low in the adult cerebellum to affect receptor levels especially in the Purkinje neurons layer, or perhaps specificity and sensitivity of binding to VLDLR or ApoER2 are different in vivo in the context of a cholesterol trafficking defect.
While further studies could be undertaken to characterize the molecular effects in the brain, we have decided that in the context of NPC1 disease outcome, this would not be informative. We present this work here to allow colleagues in the field to assess this aspect of NPC1 and conclude as we do that PCSK9 does not play a significant role in NPC1 disease.

Animal Maintenance
All animal work was reviewed and approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Animal Care and Use Committee under protocol #18-002 (28 May 2018). Heterozygous Npc1 +/mice (BALB/cNctr-Npc1m1N/J) and B6;129S6-Pcsk9 tm1Jdh/J (Pcsk9 -/-) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Npc1 +/mice were crossed with Pcsk9 -/mice to obtain double heterozygous mutants (Npc1 +/-/Pcsk9 +/-). These mice were then inter-crossed to generate double mutant mice (Npc1 -/-/Pcsk9 -/-) and the corresponding control mice. Pups were weaned 3 weeks post-birth and subsequently had free access to food and water. Genotypes were confirmed by PCR using DNA extracted from ear clips and the genotyping protocol reported by The Jackson Laboratory (protocol ID=27858). A humane end of life timepoint was determined in conjunction with the animal facility veterinarian, by assessing severity of motor impairment, at which point the mice were euthanized and tissues analyzed as necessary.

Disease Severity Scoring
After weaning, mice were assessed weekly for weight and disease severity using a standardized scoring system modified for NPC1 [46]. The disease severity score consisted of 5 components: Grooming, gait, kyphosis, hindlimb clasp, and ledge test, with each observation scored from 0-3; 0 being the least severe and 3 indicating a severely affected phenotype. Upon entry to the procedure room, mice were allowed to acclimate to the new environment for 15 minutes. For the assessment, a mouse was randomly selected and weighed. The mouse was then placed into a new cage and assessed for disease phenotype. A composite disease severity score was calculated by adding the scores for each assessment. Evaluations were performed by two observers and mouse identification was revealed post-assessment. For the ledge test, mice were placed on the ledge of the cage and assessed for their ability to walk around the ledge with coordinated use of hindlimbs, proper balance, and ability to dismount effortlessly.

AccuScan Activity Assessment
One of the measures of motor coordination is the ability of the mice to rear up on their hind legs. We therefore measured the rearing activity of the mice in a 400 mm X 400 mm AccuScan Activity box (AccuScan Instruments Inc., Columbus, OH, USA), weekly from 5-10 weeks of age. This box contains 16 infra-red laser beams that cross the area of the box at 1-inch intervals and 1 inch above the floor to measure horizontal activity. An additional set of 16 lasers are active at 3.5 inches above the floor to measure vertical activity. Mice were initially brought to the behavioral room and allowed to acclimate for 30 minutes. In the room, ambient light and sound were constant and the researcher remained silent during data acquisition. Individual mice were placed in the chambers and activity was measured automatically by beam break counts collected by computer. Horizontal and vertical counts were recorded for three contiguous 5-minute periods. Total movements were calculated and reported for the combined second and third 5-minute periods (10 minutes).

Serum Liver Enzymes and Cholesterol Analyses
To assess liver function, mice were euthanized by CO 2 and blood obtained by cardiac puncture. The samples were allowed to coagulate at room temperature for 45 min and then centrifuged for 10 min at 2000 rpm, 4 • C. The serum was saved at -20 • C until sent for analysis. Sera from control and affected mice (7-8 weeks of age) were sent to the Department of Laboratory Medicine, Clinical Center, NIH for hepatic and lipid panel screens. The liver enzymes assayed were alkaline phosphatase (AP), alanine transaminase (ALT), and aspartate transaminase (AST) using the following Roche Diagnostics reagent kits; AP #03333752 190; ALT #20764957 322; AST #20764949 322 and the lipids analyzed were total cholesterol using reagent kit #03039773 190. All assays were analyzed by Roche/Hitachi cobas c 311/501 analyzers (Roche Diagnostics GmbH, Mannheim, Germany).

Statistical Analysis
All statistical analyses were performed using the GraphPad PRISM software, version 8.3.0 (San Diego, CA, USA, www.graphpad.com). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test. The log rank Mantle-Cox test for significance was used for the Kaplan-Meyer survival curve. Statistical significance was set to p < 0.05.

Acknowledgments:
We would like to express our thanks to the staff of the NICHD animal facility for their technical assistance and animal husbandry.

Conflicts of Interest:
The authors declare no conflict of interest.