Vitamin D3 deficiency and osteopenia in spastic paraplegia type 5 indicate impaired bone homeostasis.

Hereditary spastic paraplegia type 5 (SPG5) is an autosomal recessively inherited movement disorder characterized by progressive spastic gait disturbance and afferent ataxia. SPG5 is caused by bi-allelic loss of function mutations in CYP7B1 resulting in accumulation of the oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol in serum and cerebrospinal fluid of SPG5 patients. An effect of 27- hydroxycholesterol via the estrogen and liver X receptors was previously shown on bone homeostasis. This study analyzed bone homeostasis and osteopenia in 14 SPG5 patients as a non-motor feature leading to a potential increased risk for bone fractures. T-Scores in CT bone density measurements were reduced, indicating osteopenia in SPG5 patients. Further, we analyzed various metabolites of bone homeostasis by ELISA in serum samples of these patients. We identified a lack of vitamin D3 metabolites (Calcidiol and Calcitriol), an increase in Sclerostin as a bone formation/mineralization inhibiting factor, and a decrease in cross-linked N-telopeptide of type I collagen (NTX), a marker indicating reduced bone resorption. As statin treatment has been found to lower oxysterol levels, we evaluated its effect in samples of the STOP-SPG5 trial and found atorvastatin to normalize the increased sclerostin levels. In summary, our study identified osteopenia as a non-motor feature in SPG5 and suggests the need for vitamin D3 substitution in SPG5 patients. Sclerostin may be considered a therapeutic target and biomarker in upcoming therapeutical trials in SPG5.

Metabolic profiling in serum, cerebrospinal fluid, and brain of patients with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is caused by autosomal recessive loss-of-function mutations in CYP27A1, a gene encoding cytochrome p450 oxidase essential for bile acid synthesis, resulting in altered bile acid and lipid metabolism. Here, we aimed to identify metabolic aberrations that drive ongoing neurodegeneration in some patients with CTX despite chenodeoxycholic acid (CDCA) supplementation, the standard treatment in CTX. Using chromatographic separation techniques coupled to mass spectrometry, we analyzed 26 sterol metabolites in serum and cerebrospinal fluid (CSF) of patients with CTX and in one CTX brain. Comparing samples of drug naive patients to patients treated with CDCA and healthy controls, we identified 7α,12α-dihydroxycholest-4-en-3-one as the most prominently elevated metabolite in serum and CSF of drug naive patients. CDCA treatment substantially reduced or even normalized levels of all metabolites increased in untreated patients with CTX. Independent of CDCA treatment, metabolites of the 27-hydroxylation pathway were nearly absent in all patients with CTX. 27-hydroxylated metabolites accounted for ∼45% of total free sterol content in CSF of healthy controls but <2% in patients with CTX. Metabolic changes in brain tissue corresponded well with findings in CSF. Interestingly, 7α,12α-dihydroxycholest-4-en-3-one and 5α-cholestanol did not exert toxicity in neuronal cell culture. In conclusion, we propose that increased 7α,12α-dihydroxycholest-4-en-3-one and lack of 27-hydroxycholesterol may be highly sensitive metabolic biomarkers of CTX. As CDCA cannot reliably prevent disease progression despite reduction of most accumulated metabolites, supplementation of 27-hydroxylated bile acid intermediates or replacement of CYP27A1 might be required to counter neurodegeneration in patients with progressive disease despite CDCA treatment.

Deep mining of oxysterols and cholestenoic acids in human plasma and cerebrospinal fluid: Quantification using isotope dilution mass spectrometry.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.

Generation of the CRISPR/Cas9-mediated KIF1C knock-out human iPSC line HIHRSi003-A-1.

Bi-allelic loss-of-function mutations in the gene encoding the motor protein KIF1C are associated with Hereditary Spastic Paraplegia (HSP) type SPG58, a slowly progressive neurodegenerative motoneuron disease. The biological role of KIF1C is incompletely understood. We used a protein-based CRISPR/Cas9 genome editing approach to generate a homozygous KIF1C knock-out iPSC line (HIHRSi003-A-1) from a healthy control. This iPSC-KIF1C-/- line and the corresponding isogenic control are a useful model to study the physiological function of KIF1C and the pathophysiological consequences of KIF1C dysfunction in human disease.

Comparative Transcriptional Profiling of Motor Neuron Disorder-Associated Genes in Various Human Cell Culture Models.

Disease modeling requires appropriate cellular models that best mimic the underlying pathophysiology. Human origin and an adequate expression of the disease protein are pre-requisites that support information from a model to be meaningful. In this study we investigated expression profiles of (i) PBMCs and (ii) fibroblasts as patient derived cells as well as (iii) lymphoblasts and (iv) induced pluripotent stem cells (iPSC) as immortalized sources, and (v) iPSC-derived cortical neurons to assess their aptitude to model motor neuron diseases (MNDs) including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). We generated all five different cell types from two healthy donors and performed RNA sequencing to display expression patterns in MND-related genes. For the ten most common HSP genotypes we validated gene expression by qPCR. To verify the results on protein level, proteome analysis of fibroblasts, iPSCs and cortical neurons was performed. Depending on the specific MND gene we found largely different expression patterns. Out of 168 MND-related genes, 50 had their highest expression in iPSC-derived cortical neurons, 41 were most strongly expressed in fibroblasts, 26 in lymphoblasts, 22 in iPSCs, and 14 in PBMCs. Pathophysiologically related MNDs like HSPs associated with axonal transport deficits shared highest expression in cortical neurons. 15 MND-related genes were not detectable in any of the analyzed cell types. This may reflect the critical dependency of motor neurons on support of other cell types like oligodendrocytes which express myelin proteins like L1CAM (SPG1), PLP1 (SPG2) and MAG (SPG75) which are lacking in neurons but cause MNDs if mutated. This study provides comprehensive information on expression of genes associated with a large spectrum of MNDs. Expression profiles can be used to inform on appropriate cell models for genotype specific motor neuron research.

Decreased Na+/K+ ATPase Expression and Depolarized Cell Membrane in Neurons Differentiated from Chorea-Acanthocytosis Patients.

Loss of function mutations of the chorein-encoding gene VPS13A lead to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with accelerated suicidal neuronal cell death, which could be reversed by lithium. Chorein upregulates the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the Na+/K+-ATPase, a pump required for cell survival. To explore whether chorein-deficiency affects Na+/K+ pump capacity, cortical neurons were differentiated from iPSCs generated from fibroblasts of ChAc patients and healthy volunteers. Na+/K+ pump capacity was estimated from K+-induced whole cell outward current (pump capacity). As a result, the pump capacity was completely abolished in the presence of Na+/K+ pump-inhibitor ouabain (100 µM), was significantly smaller in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (24 hours 2 mM). The effect of lithium was reversed by SGK1-inhibitor GSK650394 (24 h 10 µM). Transmembrane potential (Vm) was significantly less negative in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (2 mM, 24 hours). The effect of lithium on Vm was virtually abrogated by ouabain. Na+/K+ α1-subunit transcript levels and protein abundance were significantly lower in ChAc neurons than in control neurons, an effect reversed by lithium treatment (2 mM, 24 hours). In conclusion, consequences of chorein deficiency in ChAc include impaired Na+/K+ pump capacity.

mRNA as a Novel Treatment Strategy for Hereditary Spastic Paraplegia Type 5.

Hereditary spastic paraplegia type 5 is a neurodegenerative disease caused by loss-of-function mutations in the CYP7B1 gene encoding the oxysterol 7-α-hydroxylase involved in bile acid synthesis in the liver. Lack of CYP7B1 leads to an accumulation of its oxysterol substrates, in particular 25-hydroxycholesterol and 27-hydroxycholesterol that are able to cross the blood-brain barrier and have neurotoxic properties. A potential therapeutic strategy for SPG5 is the replacement of CYP7B1 by administration of mRNA. Here, we studied the intravenous application of formulated mouse and human CYP7B1 mRNA in mice lacking the endogenous Cyp7b1 gene. A single-dose injection of either mouse or human CYP7B1 mRNA led to a pronounced degradation of oxysterols in liver and serum within 2 days of treatment. Pharmacokinetics indicate a single injection of human CYP7B1 mRNA to be effective in reducing oxysterols for at least 5 days. Repetitive applications of mRNA were safe for at least 17 days and resulted in a significant reduction of neurotoxic oxysterols not only in liver and serum but also to some extent in the brain. Our study highlights the potential to use mRNA as a novel therapy to treat patients with SPG5 disease.

Establishment of STUB1/CHIP mutant induced pluripotent stem cells (iPSCs) from a patient with Gordon Holmes syndrome/SCAR16.

STUB1/CHIP is a central component of cellular protein homeostasis and interacts with key proteins involved in the pathogenesis of many neurodegenerative diseases. Here, we reprogrammed human skin fibroblasts from a 12-year-old male patient with recessive spinocerebellar ataxia type 16 (OMIM #615768), carrying compound heterozygous mutations (c.355C>T, c.880A>T) in STUB1. Genomic integrity of the iPSC line HIHCNi001-A without transgene integration and genomic aberration but with maintained disease-relevant mutations was proven by SNP array analysis and Sanger sequencing while pluripotency was verified by the expression of important pluripotency markers and the capacity to differentiate into cells of all three germ layers.

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

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.

Lithium Sensitive ORAI1 Expression, Store Operated Ca2+ Entry and Suicidal Death of Neurons in Chorea-Acanthocytosis.

Chorea-Acanthocytosis (ChAc), a neurodegenerative disorder, results from loss-of-function-mutations of chorein-encoding gene VPS13A. In tumour cells chorein up-regulates ORAI1, a Ca2+-channel accomplishing store operated Ca2+-entry (SOCE) upon stimulation by STIM1. Furthermore SOCE could be up-regulated by lithium. The present study explored whether SOCE impacts on neuron apoptosis. Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. ORAI1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, as well as apoptosis from annexin-V-binding and propidium-iodide uptake determined by flow cytometry. As a result, ORAI1 and STIM1 transcript levels and protein abundance and SOCE were significantly smaller and the percentage apoptotic cells significantly higher in ChAc neurons than in control neurons. Lithium treatment (2 mM, 24 hours) increased significantly ORAI1 and STIM1 transcript levels and protein abundance, an effect reversed by inhibition of Serum & Glucocorticoid inducible Kinase 1. ORAI1 blocker 2-APB (50 µM, 24 hours) significantly decreased SOCE, markedly increased apoptosis and abrogated the anti-apoptotic effect of lithium. In conclusion, enhanced neuronal apoptosis in ChAc at least partially results from decreased ORAI1 expression and SOCE, which could be reversed by lithium treatment.

Generation of induced pluripotent stem cells (iPSCs) from a hereditary spastic paraplegia patient carrying a homozygous R486C mutation in CYP7B1 (SPG5).

Skin fibroblasts were obtained from a 47-year-old hereditary spastic paraplegia patient carrying a homozygous mutation R486C in CYP7B1 (Cytochrome P450, Family 7, Subfamily B, Polypeptide 1), responsible for causing hereditary spastic paraplegia type 5 (SPG5). Induced pluripotent stem cells (iPSCs) were generated by transfection with episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated line iPS-SPG5-R486C was transgene-free, retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG5-R486C line may be a useful resource for disease modelling of SPG5.

Induced pluripotent stem cells (iPSCs) derived from cerebrotendinous xanthomatosis (CTX) patient's fibroblasts carrying a R395S mutation.

Induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts from a 60-year-old cerebrotendinous xanthomatosis (CTX) patient, carrying a homozygous mutation c. [1183C>A]; p. R395S in CYP27A1. Episomal plasmids encoding the pluripotency genes OCT4, SOX2, KLF4, L-MYC and LIN28 were introduced via electroporation. The generated line iPS-CTX-R395S has no sign of plasmid integration or chromosomal aberration and retained the mutation site in CYP27A1. Furthermore, iPSCs express pluripotency markers and are able to differentiate in all germ layers in vitro. The generated line may be a useful tool for disease modelling of CTX.

Generation of induced pluripotent stem cells (iPSCs) from a hereditary spastic paraplegia patient carrying a homozygous Y275X mutation in CYP7B1 (SPG5).

Skin fibroblasts were obtained from a 47-year-old hereditary spastic paraplegia patient carrying a homozygous mutation Y275X in CYP7B1 (Cytochrome P450, Family 7, Subfamily B, Polypeptide 1), responsible for causing hereditary spastic paraplegia type 5 (SPG5). Induced pluripotent stem cells (iPSCs) were generated by transfection with episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated line iPS-SPG5-Y275X was transgene-free, retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG5-Y275X line may be a useful resource for disease modelling of SPG5.

Membrane-anchored Aß accelerates amyloid formation and exacerbates amyloid-associated toxicity in mice.

Pathological, genetic, and biochemical hallmarks of Alzheimer's disease (AD) are linked to amyloid-ß (Aß) peptide aggregation. Especially misfolded Aß42 peptide is sufficient to promote amyloid plaque formation. However, the cellular compartment facilitating the conversion of monomeric Aß to aggregated toxic Aß species remains unknown. In vitro models suggest lipid membranes to be the driving force of Aß conversion. To this end, we generated two novel mouse models, expressing either membrane-anchored or nonanchored versions of the human Aß42 peptide. Strikingly, membrane-anchored Aß42 robustly accelerated Aß deposition and exacerbated amyloid-associated toxicity upon crossing with Aß precursor protein transgenic mice. These in vivo findings support the hypothesis that Aß-membrane interactions play a pivotal role in early-onset AD as well as neuronal damage and provide evidence to study Aß-membrane interactions as therapeutic targets.