Hereditary spastic paraplegia type 5: natural history, biomarkers and a randomized controlled trial

Abstract

Spastic paraplegia type 5 (SPG5) is a rare subtype of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative disorders defined by progressive neurodegeneration of the corticospinal tract motor neurons. SPG5 is caused by recessive mutations in the gene CYP7B1 encoding oxysterol-7α-hydroxylase. This enzyme is involved in the degradation of cholesterol into primary bile acids. CYP7B1 deficiency has been shown to lead to accumulation of neurotoxic oxysterols. In this multicentre study, we have performed detailed clinical and biochemical analysis in 34 genetically confirmed SPG5 cases from 28 families, studied dose-dependent neurotoxicity of oxysterols in human cortical neurons and performed a randomized placebo-controlled double blind interventional trial targeting oxysterol accumulation in serum of SPG5 patients. Clinically, SPG5 manifested in childhood or adolescence (median 13 years). Gait ataxia was a common feature. SPG5 patients lost the ability to walk independently after a median disease duration of 23 years and became wheelchair dependent after a median 33 years. The overall cross-sectional progression rate of 0.56 points on the Spastic Paraplegia Rating Scale per year was slightly lower than the longitudinal progression rate of 0.80 points per year. Biochemically, marked accumulation of CYP7B1 substrates including 27-hydroxycholesterol was confirmed in serum (n = 19) and cerebrospinal fluid (n = 17) of SPG5 patients. Moreover, 27-hydroxycholesterol levels in serum correlated with disease severity and disease duration. Oxysterols were found to impair metabolic activity and viability of human cortical neurons at concentrations found in SPG5 patients, indicating that elevated levels of oxysterols might be key pathogenic factors in SPG5. We thus performed a randomized placebo-controlled trial (EudraCT 2015-000978-35) with atorvastatin 40 mg/day for 9 weeks in 14 SPG5 patients with 27-hydroxycholesterol levels in serum as the primary outcome measure. Atorvastatin, but not placebo, reduced serum 27-hydroxycholesterol from 853 ng/ml [interquartile range (IQR) 683-1113] to 641 (IQR 507-694) (-31.5%, P = 0.001, Mann-Whitney U-test). Similarly, 25-hydroxycholesterol levels in serum were reduced. In cerebrospinal fluid 27-hydroxycholesterol was reduced by 8.4% but this did not significantly differ from placebo. As expected, no effects were seen on clinical outcome parameters in this short-term trial. In this study, we define the mutational and phenotypic spectrum of SPG5, examine the correlation of disease severity and progression with oxysterol concentrations, and demonstrate in a randomized controlled trial that atorvastatin treatment can effectively lower 27-hydroxycholesterol levels in serum of SPG5 patients. We thus demonstrate the first causal treatment strategy in hereditary spastic paraplegia.

Keywords: SPG5; biomarker; hereditary spastic paraplegia; oxysterol; randomized controlled trial.

Figure 1

Cross-sectional and time-to-event analyses of disease severity and progression. (A) Age of onset distribution: patients with bi-allelic missense mutations had a later age of onset [median 30 years (IQR 13–37)] than those with either one [median 8 years (IQR 5–12)] or two [median 10 years (IQR 5–14)] truncating mutations (Mann-Whitney U-test, two-sided). The median age of onset in the total cohort was 13 years (IQR 6–32). Patients carrying the Arg486Cys mutation on both alleles are marked by red dots. With the exception of one case (Patient F10.1), all carriers of the homozygous Arg486Cys mutation manifested beyond the age of 30 years. Notably, all carriers of an Arg486Cys allele, which has a minor allele frequency of 0.1% in the European population while it is absent in the Latino population, were of European decent. (B) SPRS score by disease duration: disease severity as measured by the SPRS strongly correlated with disease duration in this cross-sectional cohort of SPG5 cases (n = 31). The fitted cross-sectional progression rate (SPRS points per year with the disease) is 0.56 points/year. The 95% confidence interval of the modelled fit is shaded in blue. (C) Fast walk (10 m) by SPRS: the time needed to cover a 10 m distance on a flat surface including one turn (item 3 of the SPRS) strongly correlated with the SPRS (n = 19). The best fit was reached after logarithmic transformation of the x-axis. This measure is valid only for patients that are still able to walk and was accordingly not applicable in three cases (Patients F1.1, F14.1 and F14.2). (D) Ten steps of stairs by SPRS: the time needed to navigate 10 steps including one turn with or without support of the banister (item 5 of the SPRS) strongly correlated with the SPRS (n = 19). The best fit was reached after logarithmic transformation of the x-axis. This measure was available only for cases still able to walk steps and therefore not applicable in three cases (Patients F1.1, F14.1 and F14.2). (E) Loss of independent ambulation: Kaplan-Meier analysis indicating the probability of SPG5 cases to become dependent on a walking aid (blue line). The median disease duration until SPG5 cases depend on a walking aid is 23 years (n = 23). (F) Wheelchair dependency: Kaplan-Meier analysis indicating the probability of SPG5 cases to become dependent on a wheelchair (blue line). The median disease duration until SPG5 cases depend on a wheelchair is 33 years (n = 23). (G) Mutation spectrum in SPG5. Top: Structure of the CYP7B1 protein: The CYP7B1 gene encodes the 506 amino acid protein hydroxycholesterol 7-α-hydroxylase (NP_004811). Several alpha helices are predicted for CYP7B1 (Cui et al., 2013) (marked by grey boxes). Predicted active site residues are labelled by red circles; the heme binding site (Cys449) is marked by an asterisk. Bottom: Frequency of mutations in SPG5. The histogram shows the frequency of published mutations including this study in SPG5 families. Truncating mutations (nonsense, frameshift, splice) are depicted in red, missense mutations and inframe insertions/deletions in blue. There are several mutational hotspots in SPG5; the amino acid residues most commonly affected by missense mutations are the active site residue Arg486 (16 families), Arg417 (nine families) and Thr297 (eight families).

Figure 2

Longitudinal disease progression rate, factors influencing disease severity and oxysterol association with clinical parameters. (A) Longitudinal follow-up examinations in 21 SPG5 patients over a median follow-up interval of 12.4 months. The superimposed dotted grey line indicates the prospective progression rate of 0.80 SPRS points per year, derived from a linear mixed model with SPRS score as dependent variable and disease duration at examination as fixed effect. (B) Levels of 27-OHC (in serum) in dependence on the mutation type. Patients that carry missense mutations on both alleles have lower levels of 27-OHC in serum than cases with mono- or bi-allelic truncating mutations. Blue lines indicate median 27-OHC levels in serum. (C and D) Higher levels of 27-OHC in serum are associated with more severe disease and explain 36% of the variability of the SPRS score (P = 0.007). In cross-sectional data obtained from 19 SPG5 patients serum 27-OHC levels are furthermore higher at later disease stages (r²= 0.33; P = 0.010). (E) Disease severity increases with longer disease durations. Groups ‘short’, ‘medium’ and ‘long’ correspond to subgroups of the total cohort classified into tertiles (n = 19). (F) No differences of disease severity are observed depending on age of onset tertiles when considering the total cohort (n = 19). (G) In the subgroups with medium and long disease duration, later onset is associated with more severe disease (n = 19).

Figure 3

Neurotoxicity of oxysterols. The motor neuron-like cell line NSC-34 (A and B) as well as cortical neurons derived from iPSCs (C and D) were exposed to increasing concentrations of 24S-OHC, 25-OHC, 27-OHC and 3β-CA. (A and C) Metabolic activity was assessed by measuring the cleavage of the tetrazolium salt WST-1 in a colorimetric assay. (B and D) LDH-release in the medium was analysed as a measure of cytotoxicity. The range of oxysterol concentration in the serum of healthy controls is indicated by shaded green boxes. Shaded red boxes show the range of values observed in SPG5 patients. Dotted grey lines are fitted following a linear regression model. The horizontal axis is log-scaled.

Figure 4

Individual treatment responses. Individual levels of cholesterol and oxysterols before and after treatment with placebo (left column) or atorvastatin (right column). Data from serum analyses are given in (A and C) and for CSF in D. The mean change of values across the group and the P-value for a sign rank test is given for each analyte. The ratio between brain-derived 24S-OHC and cholesterol (B) can be used as an indirect indicator of station effects on brain cholesterol metabolism (Thelen et al., 2006).

Figure 5

Oxysterol flux across the blood–brain barrier. 27-OHC is produced both in the periphery and the CNS by 27α-hydroxylation of cholesterol. A net flux of 27-OHC from the circulation to the CNS has been demonstrated (Heverin et al., 2005), that is most likely driven by the concentration gradient between blood and CSF (∼150 ng/ml 27-OHC in serum versus ∼0.5 ng/ml 27-OHC in CSF). To eliminate 27-OHC from the CNS, it is metabolized to 7α-hydroxy-3-oxo-4-cholestenoic acid (7α-OH-4-CA), requiring the action of three key enzymes: sterol 27-hydroxylase (CYP27A1), oxysterol 7α-hydroxylase (CYP7B1), and 3β-hydroxy-C27-steroid dehydrogenase/isomerase (HSD3B7). 7α-OH-4-CA is transferred efficiently across the blood–brain barrier (Meaney et al., 2007). However, the flux of 7α-OH-4-CA from the brain is too low as to fully explain the elimination of 27-OHC from the CNS (Meaney et al., 2007). Further routes to metabolize and excrete 27-OHC to the circulation therefore have to be assumed (see ‘metabolite X’ in the figure). BBB = blood–brain barrier.