Endogenous and synthetic neurosteroids in treatment of Niemann-Pick Type C disease

Abstract

The functions for neurosteroids during development and in response to nervous system injury are beginning to be identified. We focused on a mouse model in which we believed neurosteroid production would be altered, and which had a neurodegenerative phenotype. Niemann-Pick Type-C (NP-C) is an autosomal recessive neurodegenerative disease caused by mutations in NPC1 (95%) or NPC2 (5%), resulting in lysosomal accumulation of unesterified cholesterol and glycolipids. The NIH mouse model of NP-C has a mutation in the NPC1 gene, and exhibits several pathological features of the most severe NP-C patients. How lysosomal storage and trafficking defects lead to neurodegeneration is unknown. We found that these mice had normal neurosteroidogenic enzyme activity during development, but lost this activity in the early neonatal period, prior to onset of neurological symptoms. Neurons that expressed P450scc, 3beta HSD, as well as those that expressed 3alpha HSD and 5alpha reductase were lost in adult NP-C brains, resulting in diminished concentrations of allopregnanolone. We treated NP-C mice with allopregnanolone and found that a single dose in the neonatal period resulted in a doubling of life span, substantial delay in onset of neurological symptoms, survival of cerebellar Purkinje and granule cell neurons, and reduction in cholesterol and ganglioside accumulation. The mechanism by which allopregnanolone elicited these effects is unknown. Our in vitro studies showed that Purkinje cell survival promoted by allopregnanolone was lost by treatment with bicuculline, suggesting GABA(A) receptors may play a role. We treated NP-C mice with a synthetic GABA(A) neurosteroid, ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one). Ganaxolone treatment of NP-C mice produced beneficial neurological effects, but these effects were not as robust as those obtained using allopregnanolone. Thus, allopregnanolone may elicit its effects through GABA(A) receptors and through other mechanisms. Additional studies also suggest that allopregnanolone may elicit its effects through pregnane-X-receptors (PXR). Our data suggest that mouse models of neurodegeneration may be beneficial in establishing both physiologic and pharmacologic actions of neurosteroids. These animal models further establish the wide range of functions of these compounds, which may ultimately be useful for treatment of human diseases.

Figure 1

Effect of a single dose of allopregnanolone or ganaxolone on survival of NP-C mice. Allopregnanolone (Allo) or ganaxolone (25 mg/kg in 20% §-cyclodextrin) or nothing (untreated, 20% §-cyclodextrin only) was administered subcutaneously in a single injection at postnatal day 7. A. Survival curves for treatments. B. Average survival time. Data are means ± S.D. N=12 for untreated and allopregnanolone-treated mice; n=8 for ganaxolone-treated mice.

Figure 2

Effect of a single dose of allopregnanolone (allo) or ganaxolone on onset of symptoms of NP-C mice. A. Onset of symptoms: Animals were assessed weekly for weight, tremor, ataxia, and week of onset was noted. B. Motor coordination and C. Locomotor function: mice were assessed weekly for locomotor function and coordination. These assays used locomotor tests established for assessing spinal cord injuries and recovery (Basso, et al., 1995). The rater was blinded to treatment. Data are means ± S.D. N=12 for untreated and allopregnanolone-treated mice; n=8 for ganaxolone-treated mice.

Figure 3

Dose-responsiveness of allopregnanolone treatment. NP-C mice were treated with a single dose of allopregnanolone (0 – 50 mg/kg) at postnatal day 7. Onset of symptoms (tremor, ataxia and weight loss) was assessed weekly. Data are means ± S.D. N=12 mice for each dose.

Figure 4

RT-PCR analysis of pregnane X receptor mRNA expression in the brain. Brains from postnatal day 7 mice were treated with nothing (−ALLO) or with 25 mg/kg allopregnanolone, subcutaneously (+ALLO), and brains were removed 24 hours later. Brains were separated into various regions, RNA was isolated and cDNA was prepared and amplified with primers specific for mPXR . Amplification was for 35 cycles, and PCR products of 418 bp were separated on 2% agarose gels. Ctx, cortex; Cb, cerebellum; Hypo, hypothalamus; Thal, thalamus; Lv, liver.