Identification of Altered Developmental Pathways in Human Juvenile HD iPSC With 71Q and 109Q Using Transcriptome Profiling

Abstract

In Huntington disease (HD) subtle symptoms in patients may occur years or even decades prior to diagnosis. HD changes at a molecular level may begin as early as in cells that are non-lineage committed such as stem cells or HD patients induced pluripotent stem cells (iPSCs) offering opportunity to enhance the understanding of the HD pathogenesis. In addition, juvenile HD non-linage committed cells were previously not directly investigated in detail by RNA-seq. In the present manuscript, we define the early HD and juvenile HD transcriptional alterations using 6 human HD iPS cell lines from two patients, one with 71 CAGs and one with 109 CAG repeats. We identified 107 (6 HD lines), 198 (3 HD71Q lines) and 217 (3 HD109Q lines) significantly dysregulated mRNAs in each comparison group. The analyses showed that many of dysregulated transcripts in HD109Q iPSC lines are involved in DNA damage response and apoptosis, such as CCND1, CDKN1A, TP53, BAX, TNFRSF10B, TNFRSF10C, TNFRSF10D, DDB2, PLCB1, PRKCQ, HSH2D, ZMAT3, PLK2, and RPS27L. Most of them were identified as downregulated and their proteins are direct interactors with TP53. HTT probably alters the level of several TP53 interactors influencing apoptosis. This may lead to accumulation of an excessive number of progenitor cells and potential disruption of cell differentiation and production of mature neurons. In addition, HTT effects on cell polarization also demonstrated in the analysis may result in a generation of incorrect progenitors. Bioinformatics analysis of transcripts dysregulated in HD71Q iPSC lines showed that several of them act as transcription regulators during the early multicellular stages of development, such as ZFP57, PIWIL2, HIST1H3C, and HIST1H2BB. Significant upregulation of most of these transcripts may lead to a global increase in expression level of genes involved in pathways critical for embryogenesis and early neural development. In addition, MS analysis revealed altered levels of TP53 and ZFP30 proteins reflecting the functional significance of dysregulated mRNA levels of these proteins which were associated with apoptosis and DNA binding. Our finding very well corresponds to the fact that mutation in the HTT gene may cause precocious neurogenesis and identifies pathways likely disrupted during development.

Expanded huntingtin CAG repeats disrupt the balance between neural progenitor expansion and differentiation in human cerebral organoids

“…Indeed, studies examining neurogenesis using mouse HD stem cells have given conflicting results showing that mutant HTT both impairs neurogenesis [21] and promotes it [13,22]. Efforts to examine the impact of mutant HTT on neurodevelopment and neurogenesis using human pluripotent stem cells (hPSCs) having been similarly unclear [18,19,[23][24][25][26] (reviewed in [27]). Mutant HTT was shown to impact early ectodermal development, reflected in an altered phenotypic signature, in an in vitro model of neurulation [28].…”

“…Mutant HTT was shown to impact early ectodermal development, reflected in an altered phenotypic signature, in an in vitro model of neurulation [28]. Based on transcriptional analyses of hPSCs [26], neural progenitors [23,25], and neurons [24], a number of studies 2/26 have also suggested that mutant HTT impairs neural progenitor differentiation and delays neuronal maturation. However, assessment of cortical neurogenesis showed no effect of mutant HTT on the quantity or timeline of differentiation of deep and upper layer cortical neurons [24].…”

Recent Overview of the Use of iPSCs Huntington’s Disease Modeling and Therapy

“…Results Reference HD iPSCs-MSNs -elevated caspase activity upon growth factor deprivation [12] HD iPSC-MSN -neuroprotective effect of CGS21680 and APEC therapeutic potential [18] HD iPSC-NPCs -higher levels of FOXO1 and FOXO4 elevated proteasome activity [19] iPSC-GABA + neurons -under treatment with memantine reversal of HD pathologic events [20] HD monkey iPSC-astrocytes -detection of numerous HD related pathologies mHTT aggregates, inefficient glutamate clearance, suppression of mitochondrial function, abnormal electrophysiology [21] Corrected HD iPSC-NPCs -after transplantation into mice model survival and differentiation of cells into the GABAergic neurons [22] iPSC-NSCs -after bilateral transplantation into mice striatum improved locomotor function [33] mice HD iPSCs/human HD iPSCs -dysregulation of ERK signaling, β-catenin phosphorylation, SOD1 accumulation and p53 expression [34] Juvenile HD-iPSCs -high number of significantly dysregulated mRNAs [35] HD iPSC-MSN -increased calcium SOC activity; treatment by quinazoline derivative -EVP4593 led to reduced activity of SOC currents and normalization of calcium transport [23] HD monkey iPSC-NPCs -under treatment with memantine, Rilizole and Methylene blue the most potent anti-apoptotic drug was Rilizole; the most effective in reduction of mTT aggregates was Methylene blue [36] Corrected HD monkey iPSC-GABA + neurons -after transplantation into mice striatum longer lifespan of HD mice model; improved behavioral and locomotor function [37] Zhang et al [12] were among the first authors to generate an iPSC-derived HD model. The researchers developed iPSCs from an HD patient displaying 72 CAG repeats, which were used to generate striatal neurons susceptible to cellular damage with typical characteristics of HD, such as mHTT aggregation and decreased concentrations of glutamate transporters and BDNF.…”

“…The presence of these effects in pluripotent cells indicates that the pathological processes begin during early embryonic development. Another group also investigated early molecular pathologies in HD-iPSCs, but with the goal of identifying the pathological, transcriptional changes in juvenile HD-iPSCs, which occur during the neurodevelopment of HD [35]. These authors used the RNA-seq method to study the transcriptional profiles of 6 human juvenile HD-iPSC lines with 71 and 109 CAG repeats.…”

“…The results revealed numerous significantly dysregulated mRNAs, a large number of which are involved in DNA damage and apoptosis. Based on the prominent upregulation of the vast majority of transcripts, the authors hypothesized that this phenomenon could be related to an overall increase in the expression levels of molecules involved in the pathways essential for the early stages of embryonic development [35].…”