Speaker
Description
"Introduction
Huntington’s disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant heritability characterized by the aggregation of mutant huntingtin (mHTT) protein. HD is described by the region-specific neuronal degeneration of medium spiny neurons (MSNs) in the striatum, elevated Ca2+ signaling, and dendritic spine pathology. However, the pathway connecting disturbed Ca2+ signaling, which is suggested as an early event in HD pathology, and the neuronal death is unclear. We found that huntingtin-associated protein 1 isoform A (HAP1A), a binding partner of mHTT, dysregulates Ca2+ dynamics in the endoplasmic reticulum (ER) through increased activation of inositol-(1,4,5)triphosphate receptor 1 (IP3R1). Opening of IP3R1 via HAP1A elevates store-operated calcium entry (SOCE) in YAC128 MSN cultures, HD mouse model for in vitro studies (Czeredys et al. 2018). Enhanced SOCE activity leads to striatal synaptic loss. ER stress is also dysregulated in HD models. Therefore, we hypothesized that dysregulated (via HAP1A) Ca2+ signaling is responsible for the activation of unfolded protein response (UPR), which in turn leads to neurodegeneration. To test our hypothesis, we will use both iPSCs-based GABAergic MSNs and striatal organoids derived from HD patients’ fibroblasts from different HD onsets.
Methodology
Fibroblasts lines with different stages of Huntington’s disease progression including presymptomatic HD, early manifest HD, manifest HD onset, as well as juvenile HD and appropriate controls were cultured and SOCE was measured using single-cell Ca2+ measurements. Fibroblast lines were reprogramed to iPSCs lines using STEMCCA Lentivirus Reprogramming Kit. Established iPSCs lines were characterized using different methods including karyotyping, STR analysis, sequencing, RT-PCR, immunofluorescence, and embryonic bodies formation assay. To test the project hypothesis iPSCs lines are being differentiated into iPSCs-based GABAergic MSNs and striatal organoids (Latoszek and Czeredys, 2021).
Results
Changes in SOCE in fibroblasts from different HD onsets were observed. We found that our iPSCs lines showed alkaline phosphatase activity and normal colony morphology as well as they are mycoplasma free. We detected that the reprogramming process does not affect the karyotype of iPSCs lines. Sequence consistency of CAG repeats in the HTT gene was demonstrated both by sequencing and PCR between fibroblasts and iPSC lines. The concordance of selected STRs between iPSCs and fibroblast lines has been shown. RT-qPCR analysis and immunofluorescence revealed the expression of pluripotency markers in iPSC lines. We also obtained embryonic bodies from iPSCs lines which were characterized by the expression for markers of three germ layers by RT-PCR and immunofluorescence.
Conclusion
iPSCs-based GABAergic MSNs and striatal organoids obtained from HD patients’ fibroblasts from different HD onsets will be an important tool to study the role of abnormal Ca2+ signaling and UPR in the neurodegeneration of HD. The present work is a significant step towards this goal.
This study was supported by the National Science Centre in Poland
(grant no. 2019/33/B/NZ3/02889 to MC).
References:
Czeredys M. et al., Front Cell Neurosci 26;12:381 (2018).
Latoszek E. and Czeredys M, Front Cell Dev Biol 1;9:657337(2021)."
52354557906
