Cell line specific alterations in genes associated with dopamine metabolism and signaling in midbrain dopaminergic neurons derived from 22q11.2 deletion carriers with elevated dopamine synthesis capacity

Abstract

Microdeletions at the 22q11.2 locus are associated with increased risk for schizophrenia. Recent work has demonstrated that antipsychotic naïve 22q11.2 carriers display elevated levels of dopamine synthesis capacity (DSC) as assessed by ¹⁸F-DOPA PET imaging. While this is consistent with a role for abnormal dopamine function in schizophrenia, it is unclear what molecular changes may be associated with this neuro-imaging endophenotype, and moreover, if these alterations occur independently of clinical presentation. We therefore conducted a pilot study in which we generated human induced pluripotent stem cells (hiPSCs) from two 22q11.2 deletion carriers with elevated DSC in vivo, but distinct clinical presentations. From these and neurotypical control lines we were able to robustly generate midbrain dopaminergic neurons (mDA-neurons). We then assessed whether genes associated with dopamine synthesis, metabolism or signaling show altered expression between genotypes and further between the 22q11.2 deletion lines. Our data showed alterations in expression of genes associated with dopamine metabolism and signaling that differed between the two 22q11.2 hiPSC lines with distinct clinical presentations. This reinforces the importance of considering clinical, genetic and molecular information, when possible, when choosing which donors to generate hiPSCs from, to carry out mechanistic studies.

Keywords: Human induced pluripotent stem cells; Midbrain floor plate neural progenitor; Reprogramming; Schizophrenia; [(18)F]-DOPA PET.

Fig. 1

Generation of midbrain dopamine neurons from 22q11.2 deletion hiPSCs. (A) Schematic of the 22q11.2 CNVs and total number of coding gene affected in the hiPSC lines generated in this study. (B) Representative images of day 50 mDA-neurons generated from control and 22q11.2 deletion hiPSCs. After 50 days of differentiation mDA-neurons are positive for the pan-neuronal marker MAP2. In addition, mDA-neurons robustly express markers of dopamine identity, including LMX1A and tyrosine hydroxylase (TH). (C) Box and whisker blots represent quantification of DAPI cells positive MAP2; and MAP2 cells positive for LMX1A and TH in DA-neurons generated from control hiPSC lines (CTR_M3, CTM_127 and CTF_007) and 22q11 deletion hiPSC lines (22DF_191(−) and 22DM_287(+)). Error bars are represented as minimum and maximum values; each data point represents technical repeats from 3 independent experiments for each hiPSC line. (D & E) Quantitative PCR (qPCR) analysis of day 50 DA-neurons demonstrates expression of key genes inferring generation of midbrain dopaminergic neurons (TH, LMX1A, NURR1, ASCL1, GRIK2, PITX3 and FOXA2). Comparisons between groups (hiPSC lines) was made using ANOVA with Bonfoerroni post-hoc analysis; n = 3 independent experiments per hiPSC line; error bars are represented as ±sem.

Fig. 2

Expression of genes associated with dopamine synthesis, metabolism and signaling in mDA-neurons generated from control and 22q11.2 deletion hiPSCs. (A) Heatmap of expression values of genes assessed in PCR array clustered based on 2^ΔΔCT values. Data for all three control lines (CTR_M3, CTM_127 and CTF_007) were combined, whereas data for each 22q11.2 deletion line (22DF_191(−) and 22DM_287(+)) were assessed separately. Gene expression values were normalised to 5 housekeeping genes. (B) Normalised expression of TH and COMT in day 50 DA-neurons generated from control hiPSC lines and 22q11 deletion hiPSC lines. (C) Normalised expression of gene associated with dopamine synthesis and metabolism in day 50 DA-neurons generated from control hiPSC lines and 22q11 deletion hiPSC lines. (D) Normalised expression of gene associated with dopamine signaling in day 50 DA-neurons generated from control hiPSC lines and 22q11 deletion hiPSC lines. (E) Normalised expression of gene associated with dopamine targets in day 50 DA-neurons generated from control hiPSC lines and 22q11 deletion hiPSC lines. (F) Normalised expression of dopamine receptor expression in day 50 DA-neurons generated from control hiPSC lines and 22q11 deletion hiPSC lines. Comparisons between groups (hiPSC lines) was made using ANOVA with Bonfoerroni post-hoc analysis; n = 3 independent experiments per hiPSC line; error bars are represented as ±sem.