Characterization of Neurodevelopmental Abnormalities in iPSC-Derived Striatal Cultures from Patients with Huntington's Disease

Abstract

Background: Huntington's disease (HD) is an inherited neurodegenerative disease and is characterized by atrophy of certain regions of the brain in a progressive manner. HD patients experience behavioral changes and uncontrolled movements which can be primarily attributed to the atrophy of striatal neurons. Previous publications describe the models of the HD striatum using induced pluripotent stem cells (iPSCs) derived from HD patients with a juvenile onset (JHD). In this model, the JHD iPSC-derived striatal cultures had altered neurodevelopment and contained a high number of nestin expressing progenitor cells at 42 days of differentiation.

Objective: To further characterize the altered neurodevelopmental phenotype and evaluate potential phenotypic reversal.

Methods: Differentiation of human iPSCs towards striatal fate and characterization by means of immunocytochemistry and stereological quantification.

Results: Here this study demonstrates a distinct delay in the differentiation of the JHD neural progenitor population. However, reduction of the JHD aberrant progenitor populations can be accomplished either by targeting the canonical Notch signaling pathway or by treatment with HTT antisense oligonucleotides (ASOs).

Conclusions: In summary, this data is postulated to reflect a potential overall developmental delay in JHD.

Keywords: Huntington’s disease; huntingtin; iPSC; nestin; neurodegenerative disease.

Fig.1

Increased expression of nestin expressing neural progenitor cells was observed on day 42 of striatal differentiation. A) Representative images of nestin and DAPI on time points (day 0, 7, 14, 28, 42, 56, and 80). Scale bar represents 50 μm. B) Stereological quantification of nestin C) NeuroD1 D) NG2 E) Map2ab F) TuJ1 G) GFAP. Statistical analysis was performed using paired T-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Error bars represent SEM.

Fig.2

Nestin co-expression with neuronal and/or glial markers is not significantly different in HD cultures. A) Nestin co-expression levels with the neuronal (TuJ1) and glial (GFAP) marker at 42 days of differentiation. B) No nestin population is over represented in HD lines at 42 days of differentiation. Statistical analysis was performed using paired T-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Error bars represent SEM.

Fig.3

Reduction of HTT levels during differentiation reduces the HD Nestin phenotype. A) Images of and B) quantification that one week of HTT knock-down (day 35–42) using antisense oligonucleotides (ASOs) does not reduce Nestin expression in the HD cultures. C) ASO targeted knock-down of mtHtt does not significantly affect the amount of TuJ1-positive, D) or GFAP-positive cells. E) Partial knockdown of HTT during the 42 days of differentiation using ASOs showed significant reduction in nestin expressing progenitor population in the striatal cultures. F) There was no significant difference in TuJ1+ cell population in both the control and HD lines with or without the HTT knockdown. G) There was no significant difference in GFAP + cell population in both the control and HD lines with or without HTT knockdown. Statistical analysis was performed using one-way ANOVA. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Error bars represent SEM.

Fig.4

HD nesting phenotype reversed by inhibition of canonical Notch pathway. A) Immunocytochemistry of the proliferation marker Ki67. The proliferation decreases in both HD (180 repeats) and control (21 repeats) lines over increasing time points of striatal differentiation. Pictures were taken at the magnification of 20X. B) Graph shows the expression of proliferation marker Ki67 at 0, 7, 14, 28, 42, and 56 days of iPSC-derived striatal cultures. The Ki67 expression decreases over increasing time points of differentiation in both HD and control lines. C) Inhibition of canonical Notch pathway showed significant reduction in nestin expressing NPC population in both control and HD cultures. Representative images of the 1 week inhibition of canonical Notch pathway using 10 μM DAPT during striatal differentiation. D) Notch inhibition decreases percentage of nestin+ cells in all lines, but a greater percentage in HD to the level of control. E) Two of the HD lines (HD 71 and HD 180) showed significant difference in TuJ1 population F) No significant difference was observed in GFAP+. G) Images of H) and quantification of apoptotic cell death by means of TUNEL assay reveal no significant increase in cell death due to Notch inhibition. Statistical analysis was performed using paired T-test for Fig. 4 B), D) – F) and one-way ANOVA for Fig. 4 H). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Error bars represent SEM.