BackgroundSpinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein resulting in neuropathology including mutant ataxin-1... Show moreBackgroundSpinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein resulting in neuropathology including mutant ataxin-1 protein aggregation, aberrant neurodevelopment, and mitochondrial dysfunction. ObjectivesIdentify SCA1-relevant phenotypes in patient-specific fibroblasts and SCA1 induced pluripotent stem cells (iPSCs) neuronal cultures. MethodsSCA1 iPSCs were generated and differentiated into neuronal cultures. Protein aggregation and neuronal morphology were evaluated using fluorescent microscopy. Mitochondrial respiration was measured using the Seahorse Analyzer. The multi-electrode array (MEA) was used to identify network activity. Finally, gene expression changes were studied using RNA-seq to identify disease-specific mechanisms. ResultsBioenergetics deficits in patient-derived fibroblasts and SCA1 neuronal cultures showed altered oxygen consumption rate, suggesting involvement of mitochondrial dysfunction in SCA1. In SCA1 hiPSC-derived neuronal cells, nuclear and cytoplasmic aggregates were identified similar in localization as aggregates in SCA1 postmortem brain tissue. SCA1 hiPSC-derived neuronal cells showed reduced dendrite length and number of branching points while MEA recordings identified delayed development in network activity in SCA1 hiPSC-derived neuronal cells. Transcriptome analysis identified 1050 differentially expressed genes in SCA1 hiPSC-derived neuronal cells associated with synapse organization and neuron projection guidance, where a subgroup of 151 genes was highly associated with SCA1 phenotypes and linked to SCA1 relevant signaling pathways. ConclusionsPatient-derived cells recapitulate key pathological features of SCA1 pathogenesis providing a valuable tool for the identification of novel disease-specific processes. This model can be used for high throughput screenings to identify compounds, which may prevent or rescue neurodegeneration in this devastating disease. (c) 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. Show less
Daoutsali, E.; Pepers, B.A.; Stamatakis, S.; Graaf, L.M. van der; Terwindt, G.M.; Parfitt, D.A.; ... ; Roon-Mom, W.M.C. van 2023
Introduction: ADutch-type cerebral amyloid angiopathy (D-CAA) is a hereditary brain disorder caused by a point mutation in the amyloid precursor protein (APP) gene. The mutation is located within... Show moreIntroduction: ADutch-type cerebral amyloid angiopathy (D-CAA) is a hereditary brain disorder caused by a point mutation in the amyloid precursor protein (APP) gene. The mutation is located within the amyloid beta (A beta) domain of APP and leads to A beta peptide accumulation in and around the cerebral vasculature. There lack of disease models to study the cellular and molecular pathological mechanisms of D-CAA together with the absence of a disease phenotype in vitro in overexpression cell models, as well as the limited availability of D-CAA animal models indicates the need for a D-CAA patient-derived model.Methods: We generated cerebral organoids from four D-CAA patients and four controls, cultured them up to 110 days and performed immunofluorescent and targeted gene expression analyses at two time points (D52 and D110).Results: D-CAA cerebral organoids exhibited A beta accumulations, showed enhanced neuronal and astrocytic gene expression and TGF beta pathway de-regulation.Conclusions: These results illustrate the potential of cerebral organoids as in vitro disease model of D-CAA that can be used to understand disease mechanisms of D-CAA and can serve as therapeutic intervention platform for various A beta-related disorders. Show less
Daoutsali, E.; Hailu, T.T.; Buijsen, R.A.M.; Pepers, B.A.; Graaf, L.M. van der; Verbeek, M.M.; ... ; Roon-Mom, W.M.C. van 2021
Dutch-type cerebral amyloid angiopathy (D-CAA) is a monogenic form of cerebral amyloid angiopathy and is inherited in an autosomal dominant manner. The disease is caused by a point mutation in exon... Show moreDutch-type cerebral amyloid angiopathy (D-CAA) is a monogenic form of cerebral amyloid angiopathy and is inherited in an autosomal dominant manner. The disease is caused by a point mutation in exon 17 of the amyloid precursor protein (APP) gene that leads to an amino acid substitution at codon 693. The mutation is located within the amyloid beta (A beta) domain of APP, and leads to accumulation of toxic A beta peptide in and around the cerebral vasculature. We have designed an antisense oligonucleotide (AON) approach that results in skipping of exon 17, generating a shorter APP isoform that lacks part of the A beta domain and the D-CAA mutation. We demonstrate efficient AON-induced skipping of exon 17 at RNA level and the occurrence of a shorter APP protein isoform in three different cell types. This resulted in a reduction of A beta 40 in neuronally differentiated, patient-derived induced pluripotent stem cells. AON-treated wild-type mice showed successful exon skipping on RNA and protein levels throughout the brain. These results illustrate APP splice modulation as a promising therapeutic approach for D-CAA. Show less
Fulgencio-Covian, A.; Alvarez, M.; Pepers, B.A.; Lopez-Marquez, A.; Ugarte, M.; Perez, B.; ... ; Richard, E. 2020
Propionic acidemia (PA) is an inherited metabolic disease caused by mutations in the PCCA and PCCB genes. We have previously generated an induced pluripotent stem cell (iPSC) line (UAMi004-A) from... Show morePropionic acidemia (PA) is an inherited metabolic disease caused by mutations in the PCCA and PCCB genes. We have previously generated an induced pluripotent stem cell (iPSC) line (UAMi004-A) from a PA patient with the c.1218_1231del14ins12 (p.Gly407Argfs*14) homozygous mutation in the PCCB gene. Here, we report the generation of the isogenic control in which the mutation was genetically corrected using CRISPR/Cas9 technology. Off-target editing presence was excluded and the iPSCs had typical embryonic stem cell-like morphology and normal karyotype that expressed pluripotency markers and maintained their in vitro differentiation potential. Show less
Graaf, L.M. van der; Gardiner, S.L.; Tok, M.; Brands, T.; Boogaard, M.W.; Pepers, B.A.; ... ; Roon-Mom, W.M.C. van 2019
Huntington disease (HD) is an autosomal dominant, neurodegenerative disease caused by a CAG repeat expansion within the coding sequence of the HTT gene, resulting in a highly toxic protein with an... Show moreHuntington disease (HD) is an autosomal dominant, neurodegenerative disease caused by a CAG repeat expansion within the coding sequence of the HTT gene, resulting in a highly toxic protein with an expanded polyglutamine stretch that forms typical protein aggregates throughout the brain. We generated human induced pluripotent stem cells (hiPSCs) from two HD patients using non-integrating Sendai virus (SeV). The hiPSCs display a normal karyotype, express all pluripotency markers, have the same CAG repeat expansion as the original fibroblasts and are able to differentiate into the three germ layers in vitro. Show less
Daoutsali, E.; Buijsen, R.A.M.; Pas, S. van de; Jong, A. 't; Mikkers, H.; Brands, T.; ... ; Roon-Mom, W.M.C. van 2019
Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced... Show moreHereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free. Show less
Buijsen, R.A.M.; Gardiner, S.L.; Bouma, M.J.; Graaf, L.M. van der; Boogaard, M.W.; Pepers, B.A.; ... ; Roon-Mom, W.M.C. van 2018
Objectives To investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute... Show moreObjectives To investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute to novel therapeutic approaches.Methods Epigenetically regulated expression of DIO2 was explored by assessing methylation of positional CpG-dinucleotides and the respective DIO2 expression in OA-affected and macroscopically preserved articular cartilage from end-stage OA patients. In a human in vitro chondrogenesis model, we measured the effects when thyroid signalling during culturing was either enhanced (excess T3 or lentiviral induced DIO2 overexpression) or decreased (iopanoic acid).Results OA-related changes in methylation at a specific CpG dinucleotide upstream of DIO2 caused significant upregulation of its expression (β=4.96; p=0.0016). This effect was enhanced and appeared driven specifically by DIO2 rs225014 risk allele carriers (β=5.58, p=0.0006). During in vitro chondrogenesis, DIO2 overexpression resulted in a significant reduced capacity of chondrocytes to deposit extracellular matrix (ECM) components, concurrent with significant induction of ECM degrading enzymes (ADAMTS5, MMP13) and markers of mineralisation (ALPL, COL1A1). Given their concurrent and significant upregulation of expression, this process is likely mediated via HIF-2α/RUNX2 signalling. In contrast, we showed that inhibiting deiodinases during in vitro chondrogenesis contributed to prolonged cartilage homeostasis as reflected by significant increased deposition of ECM components and attenuated upregulation of matrix degrading enzymes.Conclusions Our findings show how genetic variation at DIO2 could confer risk to OA and raised the possibility that counteracting thyroid signalling may be a novel therapeutic approach. Show less
