Expression and role of nicotinic acetylcholine receptors during midbrain dopaminergic neuron differentiation from human induced pluripotent stem cells

Abstract Aim Nicotinic acetylcholine receptors (nAChRs) expressed in midbrain dopaminergic (mDA) neurons modulate mDA neuronal activity. However, their expression patterns and functional roles during mDA neuronal development remain unknown. Here, we profiled the expression and function of nAChR subtypes during mDA neuron differentiation from human induced pluripotent stem cells (hiPSCs). Methods Midbrain dopaminergic neurons were differentiated from hiPSCs using a recently developed proprietary method that replicates midbrain development. The expression patterns of developmental marker proteins were monitored during mDA neuronal differentiation using immunohistochemical analysis. Gene expression of nAChR subtypes was analyzed by reverse transcription polymerase chain reaction. Pharmacological nAChR agonists and antagonists were used to reveal the role of the α6 nAChR subunit in the differentiation of mDA neurons from hiPSCs. Results CHRNA4 expression was detected at the mDA neural progenitor stage, whereas CHRNA6 expression began during the mDA neuronal stage. CHRNA7 was expressed throughout the differentiation process, including in the undifferentiated hiPSCs. We also found that LMO3, a gene expressed in a subset of substantia nigra pars compacta (SNC) DA neurons in the midbrain, showed increased expression following nicotine treatment in a concentration‐dependent manner. Additionally, 5‐iodo A85380, a selective α6 nAChR agonist, also increased LMO3 expression in hiPSC‐derived mDA neurons, and this increase was suppressed by simultaneous treatment with bPiDi, a selective α6 nAChR antagonist. Conclusion Our findings suggest that stimulating the α6 nAChR subunit on hiPSC‐derived mDA neurons may induce neuronal maturation that is biased toward SNC DA neurons.


| INTRODUC TI ON
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that are distributed throughout entire organs, including the central nervous system. 1 nAChRs are pentameric compositions from either 10 α-subunits (α1-10) or four β-subunits (β1-4), which results in varying properties and functional diversity. 2 The α4, α6, and α7 nAChR subunits are expressed in dopaminergic (DA) neurons in the midbrain and modulate midbrain DA (mDA) neuronal activity. [3][4][5][6] Dopaminergic neurons are a major cell population in the ventral midbrain that is involved in voluntary movement, cognition, motivation, and reward. mDA neurons can be anatomically and functionally distinguished into neurons projecting from three main nuclei: the substantia nigra pars compacta (SNC), ventral tegmental area (VTA), and red nucleus. [7][8][9] SNC DA neurons project to the caudate-putamen and form the nigrostriatal pathway. Loss of SNC DA neurons is a defining pathological feature of Parkinson's disease (PD) and may contribute to tremor, rigidity, and loss of postural control. 10 Recently, mDA neurons generated from human pluripotent stem cells (PSCs) have been used as an experimental model for drug discovery and as a source of cell transplantation therapy for PD. [11][12][13][14] Moreover, the current advantageous aspects are generating either SNC or VTA DA neurons from human PSCs by controlling the signaling functions during the brain developmental process. 15,16 The LIM-only protein (LMO) is a LIM domain-containing protein family that is involved in cell fate determination during embryonic development. 17 LMO3 is a coregulator that is enriched in the SNC and may contribute to Pitx3-dependent transcription. 18 A singlecell RNA-sequencing (scRNA-seq) dataset from the human ventral midbrain during embryonic development revealed that mDA neurons can be divided into three subpopulations-DA0, DA1, and DA2-with the DA2 subpopulation specifically expressing LMO3. 19 Interestingly, the dataset also indicated that some nAChRs were expressed in DA populations during midbrain development, with CHRNA6 specifically expressed in the DA2 subpopulation, whereas CHRNA4 and CHRNA7 were broadly expressed among ventral midbrain neural populations. 19 Although nAChRs are known to be expressed during mDA neuron differentiation, the timing of this expression and the function of nAChRs are not fully understood. Here, we performed nAChR expression profiling using a model that differentiates human-induced PSCs (hiPSCs) into mDA neurons. Furthermore, we investigated the role of α6 nAChRs during mDA neuron differentiation from hiPSCs using pharmacological approaches.  20 hiPSCs were cultured following a previously described method. 21

| Induction of mDA neurons
Midbrain DA neuron induction was slightly modified from a previously described protocol. 22 Briefly, the cells were seeded at a density of 500 000 cells/cm 2 on iMatrix511-silk (Nippi, Tokyo, Within 24 h after replating, 10 μM Y27632 was added to the medium. After day 21, 10 ng/mL glial cell-derived neurotrophic factor (Peprotech), 500 μM dbcAMP (Selleck), and 1 ng/mL transforming growth factor β3 (Peprotech) were added to the medium.

| Statistical analysis
All data are shown as the mean ± standard error of the mean (SEM).
Statistical significance was determined by one-way analysis of variance followed by Tukey's post hoc test. Statistical analyses were performed using Prism 9 (GraphPad, San Diego, CA, USA).

| RE SULTS
Midbrain DA neurons were induced from hiPSCs using a simplified protocol based on a recently developed method. 22 hiPSCs generated LMX1A + and FOXA2 + mDA progenitors on day 16 ( Figure 1A,B). At this stage, SOX2 + cells (neural progenitors) were more frequently observed than DCX + cells (immature neurons) ( Figure 1C), indicating that neural progenitors are indeed induced on day 16. Furthermore, TH + neurons (DA neurons) were found on day 28 ( Figure 1D) and went on to form colony-like structures and elongated neurites on day 42 ( Figure 1E).
Gene expression analysis via RT-PCR revealed that the ventral midbrain progenitor transcription factors (TFs) LMX1A and FOXA2 were expressed on day 11. The postmitotic ventral midbrain progenitor TF NR4A2 (also known as NURR1) was subsequently expressed on day 28 ( Figure 1F). Furthermore, the SNC TF LMO3 was expressed on day 28 ( Figure 1F). The representative midbrain TFs are sequentially expressed during mDA differentiation, mimicking the expression profile of mammalian ventral midbrain development. 19,23 These data indicate that our simplified protocol induced mDA neurons that closely resembled the process of ventral midbrain development.
We then investigated the expression of nAChR subtypes, CHRNA4, CHRNA6, and CHRNA7, during mDA neuron differentiation from hiPSCs by RT-PCR ( Figure 1F). CHRNA7 was constitutively expressed across the entire differentiation process, including the undifferentiated stage ( Figure 1F). CHRNA4 was expressed on day 16, the mDA neuron precursor phase, and CHRNA6 was expressed on day 35, the mDA neuron mature stage ( Figure 1F 1 μM), a selective α6 nAChR agonist, with or without bPiDi (10 μM), an α6 nAChR antagonist. Treatments were applied from day 28 to day 42 and differentiation was analyzed by quantitative PCR. Each value is represented by the mean ± SEM (n = 3). Significance (one-way analysis of variance with Tukey's test): *p < 0.05, **p < 0.01. The raw data is shown in Table S1. hiPSCs, human induced pluripotent stem cells; mDA, midbrain dopaminergic; nAChRs, nicotinic acetylcholine receptors

| DISCUSS ION
To the best of our knowledge, this was the first study to characterize the expression pattern of the nAChR subunit genes

ACK N OWLED G M ENTS
The authors would like to thank Enago (www.enago.jp) for the English language review.

FU N D I N G I N FO R M ATI O N
The study was supported by grants-in-aid from the Private

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.

E TH I C S S TATEM ENT
Approval of the research protocols by an institutional reviewer board: