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Induced pluripotent stem cell (iPSC) modelling for the identification of mechanisms behind neurodevelopmental disorders
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Human induced pluripotent stem cells (iPSCs) have opened new possibilities to recapitulate disease mechanisms and to model disorders in vitro. In the studies presented here, iPSCs were established to model neural differentiation in Down syndrome (DS), caused by trisomy for chromosome 21 (T21); Dravet syndrome (DRS), caused by variants in the SCN1A gene; and an ataxia syndrome, caused by a variant in the NFASC gene. The major aim has been to uncover molecular and cellular mechanisms behind perturbed neurogenesis in the three disorders.

In Paper I, the analysis of transcriptomes and proteomes of the DS iPSC derived neural model revealed several perturbed gene clusters with strong temporal dynamics along neural differentiation, markedly down-regulated mitochondrial genes and a dysregulation of hub proteins. These results predict complex and genome-wide changes in T21 neural cells associated with prolonged cell cycle, reduced cell growth and a perturbed energy metabolism.

In Paper II, it was demonstrated that the transcriptional profile of iPSC based neural model system for DS was enriched for differentially methylated genes and gene families when compared to a corresponding euploid model. The differentially methylated genes were enriched for transcriptional regulation and chromatin structure, suggesting novel mechanistic links between the genomic imbalance caused by T21 and the global transcriptional dysregulation in DS.

 In Paper III, it was shown that DRS patient iPSCs differentiated into GABAergic interneurons exhibit a dysregulated epilepsy gene network as well as an altered expression of genes involved in chromatin remodelling, accompanied by abnormal electrophysiological properties and increased stress sensitivity.

In Paper IV, it was shown that neural iPSCs, established from a patient with an ataxia syndrome and a novel homozygous variant in the NFASC gene, lack a full-length neurofascin-186 important for cell adhesion. The patient derived neural iPSCs showed delayed neuronal differentiation, reduced sprouting, shorter neurites and altered electrophysiology.

The Papers I-IV show that patient derived neural iPSCs enable to identify molecular and cellular mechanisms associated with neuropathogenesis. Besides specific dysregulated pathways and cellular defects in models of three developmental disorders, with shortlists of novel candidate disease biomarkers, the results are consistent with prior data and clinical presentation of patients. The knowledge gained is of paramount importance for translation into clinical settings and a step towards development of novel therapies with the ultimate goal to alleviate symptoms of affected individuals.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. , p. 63
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1622
Keywords [en]
Induced pluripotent stem cells, Neurogenesis, Neural differentiation, Down syndrome, Dravet syndrome, Ataxia
National Category
Medical and Health Sciences
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-398620ISBN: 978-91-513-0833-3 (print)OAI: oai:DiVA.org:uu-398620DiVA, id: diva2:1376592
Public defence
2020-02-07, Room A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2020-01-17 Created: 2019-12-09 Last updated: 2020-01-17
List of papers
1. Transcriptome and Proteome Profiling of Neural Induced Pluripotent Stem Cells from Individuals with Down Syndrome Disclose Dynamic Dysregulations of Key Pathways and Cellular Functions
Open this publication in new window or tab >>Transcriptome and Proteome Profiling of Neural Induced Pluripotent Stem Cells from Individuals with Down Syndrome Disclose Dynamic Dysregulations of Key Pathways and Cellular Functions
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2019 (English)In: Molecular Neurobiology, ISSN 0893-7648, E-ISSN 1559-1182, Vol. 56, no 10, p. 7113-7127Article in journal (Refereed) Published
Abstract [en]

Down syndrome (DS) or trisomy 21 (T21) is a leading genetic cause of intellectual disability. To gain insights into dynamics of molecular perturbations during neurogenesis in DS, we established a model using induced pluripotent stem cells (iPSC) with transcriptome profiles comparable to that of normal fetal brain development. When applied on iPSCs with T21, transcriptome and proteome signatures at two stages of differentiation revealed strong temporal dynamics of dysregulated genes, proteins and pathways belonging to 11 major functional clusters. DNA replication, synaptic maturation and neuroactive clusters were disturbed at the early differentiation time point accompanied by a skewed transition from the neural progenitor cell stage and reduced cellular growth. With differentiation, growth factor and extracellular matrix, oxidative phosphorylation and glycolysis emerged as major perturbed clusters. Furthermore, we identified a marked dysregulation of a set of genes encoded by chromosome 21 including an early upregulation of the hub gene APP, supporting its role for disturbed neurogenesis, and the transcription factors OLIG1, OLIG2 and RUNX1, consistent with deficient myelination and neuronal differentiation. Taken together, our findings highlight novel sequential and differentiation-dependent dynamics of disturbed functions, pathways and elements in T21 neurogenesis, providing further insights into developmental abnormalities of the DS brain.

Keywords
Down syndrome, Induced pluripotent stem cells (iPSC), Neural differentiation, RNA sequencing, Proteome profiling
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-395428 (URN)10.1007/s12035-019-1585-3 (DOI)000486010800032 ()30989628 (PubMedID)
Funder
Swedish Research Council, 2015-02424Swedish Research Council, 2015-4870Knut and Alice Wallenberg FoundationAstraZenecaScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceThe Swedish Brain Foundation, FO2018-0100
Available from: 2019-10-23 Created: 2019-10-23 Last updated: 2019-12-09Bibliographically approved
2. DNA methylation changes in Down syndrome derived neural iPSCs uncover co-dysregulation of ZNF and HOX3 families of transcription factors
Open this publication in new window or tab >>DNA methylation changes in Down syndrome derived neural iPSCs uncover co-dysregulation of ZNF and HOX3 families of transcription factors
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Down syndrome (DS) is characterized by neurodevelopmental abnormalities caused by partial or complete trisomy of human chromosome 21 (T21). Analysis of Down syndrome brain specimens has shown global epigenetic and transcriptional changes but their interplay during early neurogenesis remains largely unknown. We differentiated induced pluripotent stem cells (iPSCs) established from two DS patients with complete T21 and matched euploid donors into two distinct neural stages corresponding to early- and mid-gestational ages.

Results: Using the Illumina Infinium 450K array, we assessed the DNA methylation pattern of known CpG regions and promoters across the genome in trisomic neural iPSC derivatives, and we identified a total of 500 stably and differentially methylated CpGs that were annotated to CpG islands of 151 genes. The genes were enriched within the DNA binding category, uncovering 37 factors of importance for transcriptional regulation and chromatin structure. In particular, we observed regional epigenetic changes of the transcription factor genes ZNF69, ZNF700 and ZNF763 as well as the HOXA3, HOXB3 and HOXD3 genes. A similar clustering of differential methylation was found in the CpG islands of the HIST1 genes suggesting effects on chromatin remodeling.

Conclusions: The study shows that early established differential methylation in neural iPSC derivatives with T21 are associated with a set of genes relevant for DS brain development, providing a novel framework for further studies on epigenetic changes and transcriptional dysregulation during T21 neurogenesis.

Keywords
Down syndrome, induced pluripotent stem cells, DNA-methylation, neurogenesis, transcription factors, gene expression
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-398619 (URN)
Funder
Swedish Research Council, 2015-02424The Swedish Brain Foundation, FO2018-0100The Swedish Brain Foundation, FO2019-0210Knut and Alice Wallenberg Foundation, Bioinformatic supportAstraZeneca
Available from: 2019-12-08 Created: 2019-12-08 Last updated: 2019-12-09
3. Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment
Open this publication in new window or tab >>Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment
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2019 (English)In: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 132, article id 104583Article in journal (Refereed) Published
Abstract [en]

Dravet syndrome (DS) is an early onset refractory epilepsy typically caused by de novo heterozygous variants in SCN1A encoding the a-subunit of the neuronal sodium channel Na(v)1.1. The syndrome is characterized by age related progression of seizures, cognitive decline and movement disorders. We hypothesized that the distinct neurodevelopmental features in DS are caused by the disruption of molecular pathways in Na(v)1.1 haploinsufficient cells resulting in perturbed neural differentiation and maturation. Here, we established DS-patient and control induced pluripotent stem cell derived neural progenitor cells (iPSC NPC) and GABAergic interneuronal (iPSC GABA) cells. The DS-patient iPSC GABA cells showed a shift in sodium current activation and a perturbed response to induced oxidative stress. Transcriptome analysis revealed specific dysregulations of genes for chromatin structure, mitotic progression, neural plasticity and excitability in DS-patient iPSC NPCs and DS-patient iPSC GABA cells versus controls. The transcription factors FOXM1 and E2F1, positive regulators of the disrupted pathways for histone modification and cell cycle regulation, were markedly up-regulated in DS-iPSC GABA lines. Our study highlights transcriptional changes and disrupted pathways of chromatin remodeling in Na(v)1.1 haploinsufficient GABAergic cells, providing a molecular framework that overlaps with that of neurodevelopmental disorders and other epilepsies.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2019
Keywords
Dravet syndrome, SCN1A, Na(v)1.1, iPSC, Neural differentiation, Neurodevelopment, Chromatin architecture
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-398427 (URN)10.1016/j.nbd.2019.104583 (DOI)000497252500015 ()31445158 (PubMedID)
Funder
Swedish Research Council, 2015-02424Swedish Research Council, 2015-02417Knut and Alice Wallenberg FoundationAstraZenecaThe Swedish Brain Foundation, FO2018-0100The Swedish Brain Foundation, FO2019-0210Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

De tre första författarna delar förstaförfattarskapet.

Available from: 2019-12-06 Created: 2019-12-06 Last updated: 2019-12-09Bibliographically approved
4. Ataxia in Patients With Bi-Allelic NFASC Mutations and Absence of Full-Length NF186
Open this publication in new window or tab >>Ataxia in Patients With Bi-Allelic NFASC Mutations and Absence of Full-Length NF186
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2019 (English)In: Frontiers in Genetics, ISSN 1664-8021, E-ISSN 1664-8021, Vol. 10, article id 896Article in journal (Refereed) Published
Abstract [en]

The etiology of hereditary ataxia syndromes is heterogeneous, and the mechanisms underlying these disorders are often unknown. Here, we utilized exome sequencing in two siblings with progressive ataxia and muscular weakness and identified a novel homozygous splice mutation (c.3020-1G > A) in neurofascin (NFASC). In RNA extracted from fibroblasts, we showed that the mutation resulted in inframe skipping of exon 26, with a deprived expression of the full-length transcript that corresponds to NFASC isoform NF186. To further investigate the disease mechanisms, we reprogrammed fibroblasts from one affected sibling to induced pluripotent stem cells, directed them to neuroepithelial stem cells and finally differentiated to neurons. In early neurogenesis, differentiating cells with selective depletion of the NF186 isoform showed significantly reduced neurite outgrowth as well as fewer emerging neurites. Furthermore, whole-cell patch-clamp recordings of patient-derived neuronal cells revealed a lower threshold for openings, indicating altered Na+ channel kinetics, suggesting a lower threshold for openings as compared to neuronal cells without the NFASC mutation. Taken together, our results suggest that loss of the full-length NFASC isoform NF186 causes perturbed neurogenesis and impaired neuronal biophysical properties resulting in a novel early-onset autosomal recessive ataxia syndrome.

Keywords
neurofascin, neuronal isoform NF186, ataxia, patient-specific induced pluripotent stem cells, neuroepithelial stem cells, neurites
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-395798 (URN)10.3389/fgene.2019.00896 (DOI)000487628800001 ()
Funder
Swedish Research Council, 2015-02424Swedish Research Council, 2017-03407Swedish Research Council, 2017-02936Swedish Foundation for Strategic Research , IB13-0074The Swedish Brain FoundationSwedish Society for Medical Research (SSMF)
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-12-09Bibliographically approved

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