SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Decreased calcium flux in Niemann-Pick type C1 patient-specific iPSC-derived neurons due to higher amount of calcium-impermeable AMPA receptors.

Niemann-Pick disease type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene, resulting mainly in the accumulation of cholesterol and the ganglioside GM2. Recently, we described accumulations of these lipids in neuronal differentiated cells derived from NPC1 patient-specific induced pluripotent stem cells (iPSCs). As these lipids are essential for proper cell membrane composition, we were interested in the expression and function of voltage-gated ion channels and excitatory AMPA receptors (AMPARs) in neurons derived from three patient-specific iPSC lines. By means of patch clamp recordings and microfluorimetric measurements of calcium (Ca2+), we examined the expression of voltage-gated ion channels and AMPARs. Cells of the three used cell lines carrying the c.1836A>C/c.1628delC, the c.1180T>C or the c.3182T>C mutation demonstrated a significantly reduced AMPA-induced Ca2+-influx, suggesting an altered expression profile of these receptors. RT-qPCR revealed a significant upregulation of mRNA for the AMPA receptor subunits GluA1 and GluA2 and western blot analysis showed increased protein level of GluA2. Thus, we conclude that the observed reduced Ca2+-influx is based on an increase of GluA2 containing Ca2+-impermeable AMPARs. An attenuated function of GluRs in neurons potentially contributes to the progressive neurodegeneration observed in NPC1 and might represent an objective in regard of the development of new therapeutic approaches in NPC1.

Differentiation of human iPSCs into VSMCs and generation of VSMC-derived calcifying vascular cells.

Vascular calcification displays a major cause of death worldwide, which involve mainly vascular smooth muscle cells (VSMCs). Since 2007, there are increasing numbers of protocols to obtain different cell types from human induced-pluripotent stem cells (iPSCs), however a protocol for calcification is missing. Few protocols exist today for the differentiation of iPSCs towards VSMCs and none are known for their calcification. Here we present a protocol for the calcification of iPSC-derived VSMCs. We successfully differentiated iPSCs into VSMCs based on a modified protocol. Calcification in VSMCs is induced by a commercial StemXVivo™ osteogenic medium. Calcification was verified using Calcein and Alizarin Red S staining or Calcium assays, and molecular analyses showed enhanced expression of calcification-associated genes. The presented method could help to study genetic risk variants, using the CRISPR/Cas technology through the introduction of Knockouts or Knockins of risk variants. Finally, this method can be applied for drug screening.

Dataset in support of the generation of Niemann-Pick disease Type C1 patient-specific iPS cell lines carrying the novel NPC1 mutation c.1180T>C or the prevalent c.3182T>C mutation - Analysis of pluripotency and neuronal differentiation.

Data presented in this article demonstrate the generation and characterization of two novel Niemann-Pick disease Type C1 (NPC1) patient-specific induced pluripotent stem cell (iPSC) lines, related to the research article Trilck et al. (Diversity of Glycosphingolipid GM2 and Cholesterol Accumulation in NPC1 Patient-Specific iPSC-Derived Neurons; Brain Res.; 2017; 1657:52-61. doi: 10.1016/j.brainres.2016.11.031). For reprogramming fibroblasts, carrying the novel homozygous mutation c.1180T>C and the prevalent homozygous mutation c.3182T>C, were used. Reprogramming into patient-specific iPSCs was induced by retroviral transduction of the transcription factors Sox2, Klf4, Oct4 and c-Myc, and confirmed according to their pluripotency. The iPSCs were subsequently differentiated into neural progenitor cells, which were terminally differentiated into functional neurons and glial cells. The generation of these cell lines provides further valuable tools to investigate pathogenic mechanism of NPC1 in human neuronal cells carrying different NPC1 mutations.

Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons.

Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. On the cellular level NPC1 mutations lead to an accumulation of cholesterol and gangliosides. As a thorough analysis of the severely affected neuronal cells is unfeasible in NPC1 patients, we recently described the cellular phenotype of neuronal cells derived from NPC1 patient iPSCs carrying the compound heterozygous mutation c.1836A>C/c.1628delC. Here we expanded the analysis to cell lines carrying the prevalent mutation c.3182T>C and the novel mutation c.1180T>C, as well as to the determination of GM2 and GM3 gangliosides in NPC1 patient-specific iPSC-derived neurons and glia cells. Immunocytochemical detection of GM2 revealed punctated staining pattern predominantly localized in neurons. Detection of cholesterol by filipin staining showed a comparable staining pattern, colocalized with GM2, indicating a deposit of GM2 and cholesterol in the same cellular compartments. Accumulations were not only restricted to cell bodies, but were also found in the neuronal extensions. A quantification of the GM2 amount by HPLC-MS/MS confirmed significantly higher amounts in neurons carrying a mutation. Additionally, these cells displayed a lowered activity of the catabolic enzyme Hex A, but not B4GALNT1. Molecular docking simulations indicated binding of cholesterol to Hex A, suggesting cholesterol influences the GM2 degradation pathway and, subsequently, leading to the accumulation of GM2. Taken together, this is the first study showing an accumulation of GM2 in neuronal derivatives of patient-specific iPSCs and thus proving further disease-specific hallmarks in this human in vitro model of NPC1.

Generation and Neuronal Differentiation of Patient-Specific Induced Pluripotent Stem Cells Derived from Niemann-Pick Type C1 Fibroblasts.

Patient-specific induced pluripotent stem cells (iPSCs) are discussed to provide a powerful tool to investigate pathological mechanisms of diseases. Moreover, such cells might be a future platform for individualized personal treatment of diseases with a broad spectrum of mutations and thus resulting in phenotypical specificities.Here, we present a protocol for the induction of induced pluripotent stem cells from patient fibroblasts with Niemann-Pick type C1 disease (NPC1). The induction is based on a retroviral system, using the "classical" transcription factors, which were described by Takahashi and colleagues in 2007. To obtain a neuronal in vitro model system of NPC1, human iPSCs were differentiated to neural progenitor cells (NPCs) and subsequently to cells of the neural lineage, namely, neurons and glial cells. iPSCs, NPCs, and terminal neuronal differentiated cells (NDCs) were characterized by means of immunocytochemistry as well as patch clamp recordings and calcium imaging to prove the functional maturation.

HES5 is a key mediator of Wnt-3a-induced neuronal differentiation.

Human neural stem/progenitor cell (hNPC)-derived neuronal progeny has been suggested as a promising cell source in a variety of neurodegenerative diseases. Understanding the underlying mechanisms that regulate neuronal differentiation is essential for efficient cell-based therapies. Wnt and Notch signaling has been shown to be crucial in this process. However, their interactions in the process of neuronal differentiation remain elusive. By using human fetal (ReNcell VM) and iPS-derived hNPCs we demonstrate that Wnt-3a immediately induced a transient HES1 upregulation and a sustained HES5 repression that was accompanied by upregulation of the proneural gene MASH1. Conversely, overexpression of HES5 resulted in reduced MASH1 expression. Remarkably, HES5 overexpression efficiently blocked Wnt-3a as well as γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT)-induced neuronal differentiation that was accompanied by a strong MASH1 downregulation thus directly linking HES5 repression/MASH1 induction to the proneurogenic effect of Wnt-3a. Stabilized ß-catenin or treatment with the specific glycogen synthase kinase 3 beta (GSK3ß) inhibitor SB-216763 failed to or only partially mimicked these effects, suggesting a GSK3ß- and ß-catenin-independent mechanism. Further, inhibition of Wnt-3a-LDL-receptor-related protein 5/6 (LRP5/6) interactions using Dickkopf-1 (Dkk-1) failed to inhibit the modulatory effect of Wnt-3a on HES1/5 and neuronal differentiation. Taken together, these data identify HES5 as a key mediator of the Wnt-3a proneurogenic effect occurring independently of the classical Wnt/ß-catenin signaling cascade thus further deciphering crosstalk mechanisms of Wnt and Notch signaling pathways regulating cell fate of hNPCs.

Niemann-Pick type C1 patient-specific induced pluripotent stem cells display disease specific hallmarks.

BACKGROUND: Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach. METHODS: We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation. RESULTS: The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient. CONCLUSIONS: We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease.