PhD Student, University of Copenhagen
Stem cell based neurodegenerative disease modeling and high-throughput pathological study
One gene affected in familial frontotemporal dementia (FTD) is the charged multivesicular body protein 2B (CHMP2B) located on chromosome 3 (FTD3). Patients display global cortical and central brain atrophies, with no apparent amyloid plaque formation or conclusive hyper-phosphorylated tau aggregates. To study the cellular and molecular events of FTD3, we have previously established a well characterized human induced pluripotent stem cells (iPSCs) disease model from patients carrying the heterozygous 31449G>C mutation in CHMP2B and isogenic gene-corrected controls generated via the CRISPR-Cas9 system with subsequent in vitro neuronal differentiation. In order to systematically decode the pathogenesis, we further integrated high-throughput RNA sequencing, mass spectrometry-based proteomic studies and metabolic assays. Intriguingly we identified several candidate genes and pathways mis-regulated, which are important in celluar organization, subcellular component transportation and neuronal development including neural transmitter transportation. In order to directly decipher impaired protein protein interactions resulting from truncated CHMP2B, homozygous inserted mutant hiPSCs were generated. We subsequently performed affinity-purification mass spectrometry analyses, revealing a list of key proteins, which lost their binding capacities due to the truncation of CHMP2B. These key proteins enabled us to directly align cellular phenotypes with molecular events, indicating that such integrated omics analysis is providing a comprehensive tool for interpreting the role of mutant CHMP2B in FTD3 pathogenesis. Strikingly, we found that several dysregulated genes and pathways, which are also, affected in other neurodegenerative disorders. These findings open up for possibilities to develop pharmaceutics targeting the underlying commonalities amongst several distinct neurodegenerative diseases.
Abstract: The truncated mutant form of the charged multivesicular body protein 2B (CHMP2B) is causative for frontotemporal dementia linked to chromosome 3 (FTD3). CHMP2B is a constituent of the endosomal sorting complex required for transport (ESCRT) and, when mutated, disrupts endosome-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3 neurons displayed defective cristae formation, accompanied by deficiencies in mitochondrial respiration and increased levels of reactive oxygen. In addition, we provide evidence for perturbed iron homeostasis, presenting an in vitro patient-specific model to study the effects of iron accumulation in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development.
Pub.: 22 Feb '17, Pinned: 10 Jun '17
Abstract: Frontotemporal dementia (FTD) is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B) on chromosome 3 (FTD3), a component of the endosomal sorting complex required for transport III (ESCRT-III). We have generated an induced pluripotent stem cell (iPSC) line of a healthy individual and inserted the CHMP2B IVS5AS G-C gene mutation into both alleles, resulting in aberrant splicing ( Skibinski et al. 2006). This human iPSC line provides an ideal model to study CHMP2B-dependent phenotypes of FTD3.
Pub.: 16 Jun '16, Pinned: 10 Jun '17
Abstract: Frontotemporal dementia (FTD) is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B) on chromosome 3 (FTD3), a component of the endosomal sorting complex required for transport III (ESCRT-III). We have generated an induced pluripotent stem cell (iPSC) line of a healthy individual and inserted the CHMP2B IVS5AS G-C gene mutation into one of the alleles, resulting in aberrant splicing ( Skibinski et al., 2006). This human iPSC line provides an ideal model to study CHMP2B-dependent phenotypes of FTD3.
Pub.: 16 Jun '16, Pinned: 10 Jun '17
Abstract: Alterations in the cellular metabolic machinery of the brain are associated with neurodegenerative disorders such as Alzheimer's disease. Novel human cellular disease models are essential in order to study underlying disease mechanisms. In the present study, we characterized major metabolic pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass spectrometry, and cellular amino acid content was quantified by high-performance liquid chromatography. Additionally, we evaluated mitochondrial function using real-time assessment of oxygen consumption via the Seahorse XF(e)96 Analyzer. Moreover, in order to validate the hiPSC-derived neurons as a model system, a metabolic profiling was performed in parallel in primary neuronal cultures of mouse cerebral cortex and cerebellum. These serve as well-established models of GABAergic and glutamatergic neurons, respectively. The hiPSC-derived neurons were previously characterized as being forebrain-specific cortical glutamatergic neurons. However, a comparable preparation of predominantly mouse cortical glutamatergic neurons is not available. We found a higher glycolytic capacity in hiPSC-derived neurons compared to mouse neurons and a substantial oxidative metabolism through the mitochondrial tricarboxylic acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U-(13)C]Glutamate and [U-(13)C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans. Interestingly, isotopic labeling in metabolites from [U-(13)C]glutamate was higher than that from [U-(13)C]glutamine. Although the metabolic profile of hiPSC-derived neurons in vitro was particularly similar to the profile of mouse cortical neurons, important differences between the metabolic profile of human and mouse neurons were observed. The results of the present investigation establish hallmarks of cellular metabolism in human neurons derived from iPSC.
Pub.: 27 Feb '17, Pinned: 10 Jun '17
Abstract: The rising prevalence of progressive neurodegenerative diseases coupled with increasing longevity poses an economic burden at individual and societal levels. There is currently no effective cure for the majority of neurodegenerative diseases and disease-affected tissues from patients have been difficult to obtain for research and drug discovery in pre-clinical settings. While the use of animal models has contributed invaluable mechanistic insights and potential therapeutic targets, the translational value of animal models could be further enhanced when combined with in vitro models derived from patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls generated using CRISPR-Cas9 mediated genome editing. The iPSCs are self-renewable and capable of being differentiated into the cell types affected by the diseases. These in vitro models based on patient-derived iPSCs provide the opportunity to model disease development, uncover novel mechanisms and test potential therapeutics. Here we review findings from iPSC-based modeling of selected neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and spinocerebellar ataxia. Furthermore, we discuss the possibilities of generating three-dimensional (3D) models using the iPSCs-derived cells and compare their advantages and disadvantages to conventional two-dimensional (2D) models.
Pub.: 06 Jun '17, Pinned: 10 Jun '17
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