Research ArticleNeurodegenerative Disease

Intracisternal cyclodextrin prevents cerebellar dysfunction and Purkinje cell death in feline Niemann-Pick type C1 disease

See allHide authors and affiliations

Science Translational Medicine  25 Feb 2015:
Vol. 7, Issue 276, pp. 276ra26
DOI: 10.1126/scitranslmed.3010101
  • Fig. 1. Age of onset and severity of ataxia in untreated NPC cats and NPC cats administered IC HPβCD.

    (A) A dose-related effect on the age of onset of ataxia was noted in NPC cats receiving IC HPβCD compared to untreated NPC cats (P < 0.05). (B to D) At 24 weeks of age, the severity of ataxia (B) and head tremor (C) was diminished in cats dosed presymptomatically; individual cats receiving 60 mg of HPβCD or more showed no ataxia or head tremor at an age when untreated NPC cats were euthanized because of disease progression. No cats receiving 15 mg of HPβCD or greater developed head tremor. At 76 weeks of age, only cats receiving 30 mg of HPβCD or greater were alive, and these cats showed mild to moderate ataxia. Cats first administered HPβCD at 16 weeks of age (postsymptomatically) showed the same onset of ataxia as untreated cats (A), with individual cats showing less ataxia and head tremor at 24 weeks of age compared to untreated NPC cats (B and D). Ataxia was graded on a 0 to 4 scale with 0, none; +1, mild ataxia; +2, moderate ataxia resulting in falling when running; +3, severe ataxia resulting in falling when walking; and +4, no longer able to stand. Head tremor was graded on a 0 to 3 scale with 0, none; +1, mild; +2, moderate; and +3, severe.

  • Fig. 2. Liver histology and biochemistry in NPC cats administered SC or IC HPβCD.

    (A) Untreated NPC cats showed severe vacuolization of hepatocyte and Kupffer cell cytoplasm that was ameliorated by HPβCD (1000 mg/kg) but was unaffected by 120 mg of IC HPβCD. All cats were 24 weeks of age. Scale bar, 100 μm. (B) High-performance thin-layer chromatography (HPTLC) of total lipids in 2-mg tissue samples. Migration in chloroform/methanol/H2O (65:25:4); stained with anisaldehyde spray. The prominent storage of cholesterol and sphingomyelin was greatly reduced in all SC-treated cats but remained unchanged with IC administration. (C) HPTLC showing a similar effect of treatment on neutral glycosphingolipids from 5-mg tissue samples after saponification of total lipids. Migration as in (B); stained with orcinol-H2SO4 spray. (D) Free sphingosine concentrations [measured by high-performance liquid chromatography (HPLC)] that were increased ~50-fold in untreated NPC cats decreased about five times after SC HPβCD treatment. For the SC graph, both cats received HPβCD (8000 mg/kg). For the IC graph, data from cats receiving 15, 30, and 60 mg were included because there was no dose effect. Normal values: 37 ± 15 pmol/mg protein (mean ± SD, n = 7). Untr’d, untreated; Norm, normal; SC8, SC4, SC1, subcutaneous, 8000, 4000, or 1000 mg/kg; IC120, IC60, intracisternal, 120 or 60 mg; Chol, unesterified cholesterol; BMP, bis(monoacylglycero)phosphate; Sph, sphingomyelin; GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb3, globotriaosylceramide.

  • Fig. 3. Calbindin and GM2 immunohistochemical staining and filipin staining of cerebellum from normal control untreated NPC cats and HPβCD-treated NPC cats.

    (Top row) Immunohistochemical calbindin labeling of Purkinje cells (PCs), whose cell body and dendritic arbor are highlighted in brown. (Middle row) Filipin labeling of unesterified cholesterol, accumulation of which is noted by white cytoplasmic staining. (Bottom row) GM2 accumulation (dark brown punctae). Normal control column depicts a full complement of PCs lacking cholesterol and GM2 storage, typical of a normal cat at 25 weeks of age. Untreated NPC column shows a large axonal spheroid (red arrow) in one of the remaining PCs within this 25-week-old NPC cat as well as abundant storage of cholesterol and GM2 gangliosides present in all remaining PCs. Treatment of an NPC1 cat with 120 mg of IC HPβCD (third column) resulted in substantial PC rescue as well as decreased cholesterol and GM2 ganglioside accumulation within PCs of this 29-week-old cat. MC, molecular cell layer; GC, granular cell layer. Scale bar, 50 μm.

  • Fig. 4. Cerebral gray matter lipids in normal control untreated NPC cats and NPC cats administered IC HPβCD.

    (A) Upper panel: HPTLC of total gangliosides (from 3-mg tissue samples), showing striking and selective reduction of GM3 and GM2 in NPC cats administered IC HPβCD; the ganglioside patterns remained essentially unchanged in NPC cats administered SC HPβCD [migration in chloroform/methanol/0.2% CaCl2 (55:45:10); stained with resorcinol-HCl spray]. Lower panel: Quantitative data (24- to 26-week-old cats; GM3 and GM2 expressed as percent of total gangliosides). IC treatment at 7.5- and 3.5-mg doses appeared less efficient at reducing GM2 and GM3 than did 15-mg or higher doses of HPβCD. (B) Lactosylceramide concentrations (HPTLC from 10-mg tissue samples) were reduced in cats treated with IC HPβCD. (C) Free sphingosine concentrations (measured by HPLC, expressed as pmol/mg protein) were normalized in all IC-treated cats. (D) HPTLC of total gangliosides (from 3-mg tissue samples) of untreated NPC cats at various ages (left) and developmental profiles of GM2 and GM3 storage (right). Normal proportions (percent of total gangliosides) are 2.0 ± 0.5% for GM2 and 2.5 ± 0.6% for GM3 (mean ± SD, n = 5).

  • Fig. 5. Pulmonary histology from 6-month-old untreated NPC cats and NPC cats administered HPβCD (8000 mg/kg SC).

    (A) In untreated NPC cats, the alveolar septa were expanded by foam cells (black arrowheads) as well as by macrophages containing larger irregular clear vacuoles. Alveolar spaces similarly contained foam cells (black arrows). (B) Cats given HPβCD (8000 mg/kg SC) had evidence of acute to subacute diffuse alveolar damage. Alveolar spaces contained abundant proteinaceous fluid with wispy strands of fibrin, foamy macrophages, and neutrophils. The alveolar septa were lined by hypertrophied type II pneumocytes with multifocal hyaline membrane formation. The septa were congested and contained foamy macrophages (black arrows), neutrophils, and lymphocytes. Arrowheads denote thickened alveolar septae. No evidence of alveolar damage was seen in the other treatment groups. (C) Lung from a normal control cat. Scale bar, 100 μm.

  • Fig. 6. Filipin staining of marginal gyrus of cerebral cortex in NPC cats.

    (A and B) Untreated 24-week-old NPC cats (B) showed storage of cholesterol (visualized as white cytoplasmic labeling within cells) in the marginal gyrus of the cerebral cortex, and no cholesterol storage was observed in healthy control animals (A). (C) Cats receiving HPβCD (1000 mg/kg SC) showed no reduction in cholesterol staining. (D) Cats receiving HPβCD (8000 mg/kg SC) showed substantially less cholesterol staining than did untreated NPC cats. Indeed, the example shown is from a 37-week-old cat that had received SC HPβCD until 24 weeks of age, at which time it was discontinued. (E and H) Cats (24 weeks old) receiving 30 mg of IC HPβCD or greater presymptomatically showed the greatest amelioration of the cholesterol storage defect. In contrast, treatment of NPC cats with 3.8 mg of IC HPβCD (G) or 120 mg of IC HPβCD administered postsymptomatically (F) revealed little effect on stored cholesterol at 24 weeks of age, whereas both 30 and 120 mg of HPβCD administered presymptomatically resulted in substantial reductions in cholesterol storage. Scale bar, 100 μm.

  • Fig. 7. Calbindin immunofluorescence of cerebellar paravermis in 6-month-old NPC cats.

    (A and B) Untreated NPC cats (B) showed a severe loss of Purkinje cells compared to normal control cats (A). (C and D) SC HPβCD administration at a dose of 8000 mg/kg resulted in increased Purkinje cell survival (D), whereas cats receiving HPβCD (1000 mg/kg SC) were indistinguishable from untreated NPC cats (C). (H) Cats treated with IC HPβCD at 30 mg or greater resulted in substantial Purkinje cell survival. In contrast, 3.8 mg of IC HPβCD (G) or 120 mg of IC HPβCD given either presymptomatically (E) or postsymptomatically (120 mg IC, late) (F) resulted in small but nonsignificant increases in Purkinje cell number compared to untreated NPC cats. Scale bar, 200 μm.

  • Fig. 8. Purkinje cell quantification and hearing threshold in NPC cats administered IC HPβCD.

    (A) Untreated 24-week-old NPC cats showed significantly fewer Purkinje cells per unit length compared to age-matched normal control cats (*P < 0.05). Cats (24 weeks old) receiving 30 mg of IC HPβCD or greater showed significantly greater Purkinje cell numbers compared to untreated NPC cats (P < 0.05). In contrast, 24-week-old cats treated postsymptomatically with 120 mg of IC HPβCD at 16 weeks of age showed significantly fewer Purkinje cells compared to normal control cats (*P < 0.05). (B) Untreated 24-week-old NPC cats showed no significant difference in hearing threshold compared to normal control cats. In contrast, cats receiving 7.5 or 120 mg of HPβCD developed significant increases in hearing threshold compared to untreated NPC cats (P < 0.05). Individual cats receiving lower doses of HPβCD also showed elevated hearing thresholds, although, as a group, these were not significant. (C) At 76 weeks of age, all NPC cats receiving 30 mg of HPβCD or greater showed significant elevations in hearing threshold. *P < 0.001.

  • Table 1. Route of administration and dose of HPβCD administered to 11 groups of cats.
    GenotypeRoute of
    administration
    DoseDosing intervalAnimal numbersSurvival time mean and
    SD, or longest (weeks)
    MalesFemales
    No mutant allele
    (normal control)
    UntreatedUntreatedUntreated2624>76*
    NPC1UntreatedUntreatedUntreated221720.7 ± 5.0
    NPC1SC1000 mg/kg and
    25 mg/kg
    (allopregnanolone)
    7 days5121.8 ± 6.5
    NPC1SC4000 mg/kg7 days0231, 36
    NPC1SC8000 mg/kg7 days3235.2 ± 12.4
    NPC1IC3.8 mg14 days1249
    NPC1IC7.5 mg14 days1262
    NPC1IC15 mg14 days2166
    NPC1IC30 mg14 days15>76*
    NPC1IC60 mg14 days21>76*
    NPC1IC120 mg14 days27>76*
    NPC1IC, SC120 mg, 1000 mg/kg14 days, 7 days53>76*
    NPC1IC120 mg postsymptomatic14 days4443.5 ± 5.8

    *No cats in this group died of NPC disease during the study period of 76 weeks. †Longest survival time of individual cats that died due to signs of NPC disease. Remaining cats in this group were euthanized at ~24 weeks of age to collect histological data. ‡Treatment first began at 16 weeks of age.

    • Table 2. Serum ALT activity and albumin and cholesterol concentrations in NPC cats.

      Mean ± SD for serum ALT, albumin, and total cholesterol of 24-week-old cats. P values are provided for groups significantly different from untreated normal control cats (*) or significantly different from untreated NPC cats (†). SD is not provided for 4000 mg/kg group, where n = 2.

      Untreated
      normal control
      Untreated
      NPC
      1000 mg/kg
      SC
      4000 mg/kg
      SC
      8000 mg/kg
      SC
      30 mg
      IC
      120 mg
      IC
      120 mg IC and
      1000 mg/kg SC
      ALT (U/liter)62.1 ± 15.4395.8 ± 169
      *P = 0.0000002
      128.1 ± 127.6
      P = 0.0003
      37.577 ± 66.9
      P = 0.00001
      295.3 ± 107
      *P = 0.003
      223.8 ± 87
      *P = 0.003
      P = 0.003
      135 ± 91
      P = 0.00004
      Albumin (g/dl)2.9 ± 0.32.6 ± 0.3
      *P = 0.00035
      2.8 ± 0.23.03.3 ± 0.38
      *P = 0.002
      P = 0.000002
      2.5 ± 0.01
      *P = 0.00002
      2.5 ± 0.2
      *P = 0.00006
      3.0 ± 0.3
      P = 0.008
      Cholesterol
      (mg/dl)
      120.3 ± 23240.3 ± 72
      *P = 0.000001
      189.9 ± 48.5
      *P = 0.004
      P = 0.04
      131166 ± 58
      P = 0.04
      191.2 ± 37.3
      *P = 0.004
      P = 0.04
      180.6 ± 22.4
      *P = 0.0001
      P = 0.004
      166 ± 34.6*
      *P = 0.007
      *P = 0.002
    • Table 3. PK of HPßCD after IC or IV administration in normal control cats.

      NC, not calculated due to below detectable concentrations for some samples.

      AnalyteRoute of
      administration
      DoseMatrixC0
      (μg/ml)
      AUC0-∞
      (μg·hour/ml)
      AUC0-24h
      (μg·hour/ml)
      t½
      (hour)
      CL/F
      (ml/hour·kg)
      Vdss
      (ml/kg)
      HPβCD (Sigma)IC120 mgCSF11,64528,6140.7–2.6
      HPβCD (Kleptose)IC30 mgCSF15,40023,30023,1003.221.294.14
      HPβCD (Kleptose)IV1000 mg/kgCSF13.1NC346NCNCNC
      HPβCD (Kleptose)IV1000 mg/kgPlasma3,7604,6606,7701.11215336

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/7/276/276ra26/DC1

      Fig. S1. Weekly mean body weights of normal control, untreated NPC, and NPC cats administered SC or IC HPβCD.

      Fig. S2. Liver gangliosides and brain glucosylceramide.

      Fig. S3. Immunofluorescence staining of GM2 ganglioside in marginal gyrus of cerebral cortex.

      Fig. S4. Immunofluorescence staining of MBP in cerebellar white matter.

      Fig. S5. Abnormal posture and MRI of shoulders of 76-week-old NPC cat that received both SC and IC HPβCD.

      Video S1. Untreated 21-week-old NPC cat showing tremor (+3), ataxia (+4), and inability to stand without support.

      Video S2. NPC cat (22 weeks old) administered HPβCD (8000 mg/kg SC) weekly since 3 weeks of age showing mild head tremor (+1) and ataxia (+1).

      Video S3. NPC cat (24 weeks old) administered 120 mg of HPβCD IC biweekly since 3 weeks of age appearing neurologically normal.

      Video S4. NPC cat (76 weeks old) administered 120 mg of HPβCD IC biweekly since 3 weeks of age showing no head tremor and mild ataxia (+1).

      Video S5. NPC cat (24 weeks old) administered 120 mg of HPβCD IC biweekly since 16 weeks of age showing head tremor (+2) and ataxia (+2).

    • Supplementary Material for:

      Intracisternal cyclodextrin prevents cerebellar dysfunction and Purkinje cell death in feline Niemann-Pick type C1 disease

      Charles H. Vite,* Jessica H. Bagel, Gary P. Swain, Maria Prociuk, Tracey U. Sikora, Veronika M. Stein, Patricia O'Donnell, Therese Ruane, Sarah Ward, Alexandra Crooks, Su Li, Elizabeth Mauldin, Susan Stellar, Marc De Meulder, Mark L. Kao, Daniel S. Ory, Cristin Davidson, Marie T. Vanier, Steven U. Walkley

      *Corresponding author. E-mail: vite@vet.upenn.edu

      Published 25 February 2015, Sci. Transl. Med. 7, 276ra26 (2015)
      DOI: 10.1126/scitranslmed.3010101

      This PDF file includes:

      • Fig. S1. Weekly mean body weights of normal control, untreated NPC, and NPC cats administered SC or IC HPβCD.
      • Fig. S2. Liver gangliosides and brain glucosylceramide.
      • Fig. S3. Immunofluorescence staining of GM2 ganglioside in marginal gyrus of cerebral cortex.
      • Fig. S4. Immunofluorescence staining of MBP in cerebellar white matter.
      • Fig. S5. Abnormal posture and MRI of shoulders of 76-week-old NPC cat that received both SC and IC HPβCD.
      • Legends for videos S1 to S5

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Video S1 (.mov format). Untreated 21-week-old NPC cat showing tremor (+3), ataxia (+4), and inability to stand without support.
      • Video S2 (.mov format). NPC cat (22 weeks old) administered HPβCD (8000 mg/kg SC) weekly since 3 weeks of age showing mild head tremor (+1) and ataxia (+1).
      • Video S3 (.mov format). NPC cat (24 weeks old) administered 120 mg of HPβCD IC biweekly since 3 weeks of age appearing neurologically normal.
      • Video S4 (.mov format). NPC cat (76 weeks old) administered 120 mg of HPβCD IC biweekly since 3 weeks of age showing no head tremor and mild ataxia (+1).
      • Video S5 (.mov format). NPC cat (24 weeks old) administered 120 mg of HPβCD IC biweekly since 16 weeks of age showing head tremor (+2) and ataxia (+2).

      [Download Video S1]

      [Download Video S2]

      [Download Video S3]

      [Download Video S4]

      [Download Video S5]

    Stay Connected to Science Translational Medicine

    Navigate This Article