Generation of the human iPSC line AKOSi010-A from fibroblasts of a female FAHN patient, carrying the compound heterozygous mutation p.Gly45Arg/p.His319Arg.

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare childhood onset neurodegenerative disease caused by mutations in the FA2H gene. Patients display abnormal myelination, cerebellar atrophy and some have iron deposition in the central nervous system. Here we describe the generation of AKOSi010-A, a human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a female patient carrying the compound heterozygous p.Gly45Arg/p.His319Arg, using non-integrating Sendai virus. The generated iPSCs express pluripotency markers, can differentiate into cell types of the three germ layers and show a normal karyotype. This cell line displays a unique source to study the pathophysiology of FAHN.

Assessment of FDA-Approved Drugs as a Therapeutic Approach for Niemann-Pick Disease Type C1 Using Patient-Specific iPSC-Based Model Systems.

Niemann-Pick type C1 (NP-C1) is a fatal, progressive neurodegenerative disease caused by mutations in the NPC1 gene. Mutations of NPC1 can result in a misfolded protein that is subsequently marked for proteasomal degradation. Such loss-of-function mutations lead to cholesterol accumulation in late endosomes and lysosomes. Pharmacological chaperones (PCs) are described to protect misfolded proteins from proteasomal degradation and are being discussed as a treatment strategy for NP-C1. Here, we used a combinatorial approach of high-throughput in silico screening of FDA-approved drugs and in vitro biochemical assays to identify potential PCs. The effects of the hit compounds identified by molecular docking were compared in vitro with 25-hydroxycholesterol (25-HC), which is known to act as a PC for NP-C1. We analyzed cholesterol accumulation, NPC1 protein content, and lysosomal localization in patient-specific fibroblasts, as well as in neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells (iPSCs). One compound, namely abiraterone acetate, showed comparable results to 25-HC and restored NPC1 protein level, corrected the intracellular localization of NPC1, and consequently decreased cholesterol accumulation in NPC1-mutated fibroblasts and iPSC-derived neural differentiated and hepatocyte-like cells. The discovered PC altered not only the pathophysiological phenotype of cells carrying the I1061T mutation- known to be responsive to treatment with PCs-but an effect was also observed in cells carrying other NPC1 missense mutations. Therefore, we hypothesize that the PCs studied here may serve as an effective treatment strategy for a large group of NP-C1 patients.

Impact of Organelle Transport Deficits on Mitophagy and Autophagy in Niemann-Pick Disease Type C.

Defective mitochondria are pathophysiological features of a number of neurodegenerative diseases. Here, we investigated mitochondrial dysfunction in the context of the rare lysosomal storage diseases Niemann-Pick disease type C1 and type C2 (NP-C1 and NP-C2). Mutations in either the NPC1 or NPC2 gene lead to cholesterol accumulation in late endosomes and lysosomes, resulting in impaired cholesterol homeostasis. The extent to which this may lead to mitochondrial dysfunction has been poorly studied so far. Therefore, we investigated the morphology, function, and transport of mitochondria, as well as their degradation via mitophagy, in a disease-associated human neural cell model of NP-C. By performing live cell imaging, we observed markedly reduced mitochondrial transport, although morphology and function were not appreciably altered. However, we observed a defective mitophagy induction shown by a reduced capability to elevate parkin expression and engulf mitochondria in autophagosomes after treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). This was accompanied by defects in autophagy induction, exhibited by a hampered p62 expression and progression, shown by increased LC3BII levels and a defective fusion of autophagosomes and lysosomes. The latter might have been additionally influenced by the observed reduced lysosomal transport. Hence, we hypothesized that a reduced recycling of mitochondria contributes to the pathophysiology of NP-C.

Patient-Specific iPSC-Derived Neural Differentiated and Hepatocyte-like Cells, Carrying the Compound Heterozygous Mutation p.V1023Sfs*15/p.G992R, Present the "Variant" Biochemical Phenotype of Niemann-Pick Type C1 Disease.

Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder caused by autosomal recessive mutations in the NPC1 gene. Patients display a wide spectrum on the clinical as well as on the molecular level, wherein a so-called "variant" biochemical phenotype can be observed. Here, we report an in vitro analysis of fibroblasts obtained from an NP-C1 patient carrying the undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R. Since NP-C1 is a neurovisceral disease and the patient suffers from severe neurological as well as hepatic symptoms, we extended our study to neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells. We detected slightly increased intracellular cholesterol levels compared to the control cell line in fibroblasts, neural differentiated and hepatocyte-like cells, suggesting a "variant" biochemical phenotype. Furthermore, the total NPC1 protein, as well as post-ER glycoforms of the NPC1 protein, tended to be reduced. In addition, colocalization analysis revealed a mild reduction of the NPC1 protein in the lysosomes. The patient was diagnosed with NP-C1 at the age of 34 years, after an initial misdiagnosis of schizophrenia. After years of mild and unspecific symptoms, such as difficulties in coordination and concentration, symptoms progressed and the patient finally presented with ataxia, dysarthria, dysphagia, vertical supranuclear gaze palsy, and hepatosplenomegaly. Genetic testing finally pointed towards an NP-C1 diagnosis, revealing the so-far undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R in the NPC1 gene. In light of these findings, this case provides support for the p.G992R mutation being causative for a "variant" biochemical phenotype leading to an adult-onset type of NP-C1 disease.

Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T.

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.

Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Niemann-Pick Type C1.

The lysosomal storage disorders Niemann-Pick disease Type C1 (NPC1) and Type C2 (NPC2) are rare diseases caused by mutations in the NPC1 or NPC2 gene. Both NPC1 and NPC2 are proteins responsible for the exit of cholesterol from late endosomes and lysosomes (LE/LY). Consequently, mutations in one of the two proteins lead to the accumulation of unesterified cholesterol and glycosphingolipids in LE/LY, displaying a disease hallmark. A total of 95% of cases are due to a deficiency of NPC1 and only 5% are caused by NPC2 deficiency. Clinical manifestations include neurological symptoms and systemic symptoms, such as hepatosplenomegaly and pulmonary manifestations, the latter being particularly pronounced in NPC2 patients. NPC1 and NPC2 are rare diseases with the described neurovisceral clinical picture, but studies with human primary patient-derived neurons and hepatocytes are hardly feasible. Obviously, induced pluripotent stem cells (iPSCs) and their derivatives are an excellent alternative for indispensable studies with these affected cell types to study the multisystemic disease NPC1. Here, we present a review focusing on studies that have used iPSCs for disease modeling and drug discovery in NPC1 and draw a comparison to commonly used NPC1 models.

Generation of an iPSC line (AKOSi004-A) from fibroblasts of a female adult NPC1 patient, carrying the compound heterozygous mutation p.Val1023Serfs*15/p.Gly992Arg and of an iPSC line (AKOSi005-A) from a female adult control individual.

Niemann-Pick disease Type C (NPC) is a rare progressive neurodegenerative disorder with an incidence of 1:120,000 caused by mutations in the NPC1 or NPC2 gene leading to a massive cholesterol accumulation. Here, we describe the generation of induced pluripotent stem cells (iPSCs) of an affected female adult individual carrying the NPC1 mutation p.Val1023Serfs*15/p.Gly992Arg and an iPSC line from an unrelated healthy female adult control individual. Human iPSCs were derived from fibroblasts using retroviruses carrying the four reprogramming factors OCT4, SOX2, KLF4 and C-MYC. These lines provide a valuable resource for studying the pathophysiology of NPC and for pharmacological intervention.

Generation of two induced pluripotent stem cell lines from a female adult homozygous for the Wilson disease associated ATP7B variant p.H1069Q (AKOSi008-A) and a healthy control (AKOSi009-A).

Wilson disease (WD) is a rare, monogenic disorder caused by mutations in the gene ATP7B. A loss of function of the expressed protein leads to excessive hepatic and cerebral copper storage. In this study, we present the generation of two induced pluripotent stem cell (iPSC) lines derived from fibroblasts of a clinically asymptomatic, chelator treated female WD patient carrying the common missense mutation p.H1069Q and an age-matched female healthy control subject. The generated iPSC lines expressed pluripotency markers, showed differentiation potential and retained their parental genotype. Therefore, these cells provide a valuable resource to understand the pathophysiology of WD and can be used as model systems for drug testing.

Oxidative Stress and Alterations in the Antioxidative Defense System in Neuronal Cells Derived from NPC1 Patient-Specific Induced Pluripotent Stem Cells.

Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer's disease and Parkinson's disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.

Generation of an iPSC line (AKOSi006-A) from fibroblasts of an NPC1 patient, carrying the homozygous mutation p.I1061T (c.3182 T > C) and a control iPSC line (AKOSi007-A) using a non-integrating Sendai virus system.

Niemann-Pick disease type C1 (NPC1) is a rare inherited lipid storage disorder caused by mutations in the NPC1 gene. Mutations lead to impaired lipid trafficking and subsequently to accumulation of cholesterol and sphingolipids. NPC1-patients present variable multisystemic symptoms, including neurological deficits. Here, we describe the generation of human iPSC lines obtained from fibroblasts of a male individual, carrying the homozygous mutation p.I1061T, and an unrelated and healthy male individual. A non-integrating Sendai virus system, containing KLF4, OCT3/4, SOX2 and C-MYC, was used for reprogramming. These cell lines provide a valuable resource for studying the pathophysiology of multisystemic NPC1-disease.

Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Niemann-Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1-/- mice and evaluated specific effects of treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1-/- mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1-/- mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

Generation of induced pluripotent stem cell lines AKOSi002-A and AKOSi003-A from symptomatic female adults with Wilson disease.

Wilson disease (WD) is an inherited, autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene. Pathogenic single nucleotide variants (SNVs) lead to functional impairment of the copper transporting ATPase ATP7B, resulting in copper accumulation and toxicity in the liver and brain. We describe the generation of two induced pluripotent stem cell (iPSC) lines derived from fibroblasts of two female WD patients. Patient 1 is compound heterozygous for p.E1064A and p.H1069Q. Patient 2 is homozygous for p.M769V. These iPSCs represent a WD model for pathophysiological studies and pharmacological screening.

Generation of the Niemann-Pick type C2 patient-derived iPSC line AKOSi001-A.

Niemann-Pick disease Type C (NPC) is a rare progressive neurodegenerative disorder with an incidence of 1:120,000 caused by mutations in the NPC1 or NPC2 gene. Only 5% of NPC patients suffer from mutations of the NPC2 gene. Here we demonstrate the generation of a Niemann-Pick disease Type C2 (NPC2) patient-derived induced pluripotent stem cell line. This cell line is capable to differentiate into derivatives of the neuronal lineage, providing a valuable tool to study pathogenic mechanisms of NPC2.

Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test.

Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous, multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study was to establish a test that allows for an objective assessment of the pathological potential of NPC1 gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying distinct sequence variants. Filipin staining was used to test for complementation of the phenotype. The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and 24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met, and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance. Moreover, we present a system that can be utilized to screen for new drugs.