α7 nicotinic acetylcholine receptors regulate radial glia fate in the developing human cortex

Abstract

Prenatal nicotine exposure impairs fetal cortical grey matter volume, but the precise cellular mechanisms remain poorly understood. This study elucidates the role of nicotinic acetylcholine receptors (nAChRs) in progenitor cells and radial glia (RG) during human cortical development. We identify two nAChR subunits-CHRNA7 and the human-specific CHRFAM7A-expressed in SOX2+ progenitors and neurons, with CHRFAM7A particularly enriched along RG endfeet. nAChR activation in organotypic slices and dissociated cultures increases RG proliferation while decreasing neuronal differentiation, whereas nAChR knockdown reduces RG and increases neurons. Single-cell RNA sequencing reveals that nicotine exposure downregulates key genes in excitatory neurons (ENs), with CHRNA7 or CHRFAM7A selectively modulating these changes, suggesting an evolutionary divergence in regulatory pathways. Furthermore, we identify YAP1 as a critical downstream effector of nAChR signaling, and inhibiting YAP1 reverses nicotine-induced phenotypic alterations in oRG cells, highlighting its role in nicotine-induced neurodevelopmental pathophysiology.

Fig. 1. Acetylcholinesterase (AChE) is expressed in the developing prefrontal cortex throughout cortical development (GW15-22).

a Representative images showing immunostaining of AChE+ cholinergic fibers, HOPX+ outer radial glia and vimentin (VIM)+ radial glia fibers at GW15, and GW22. Scale bar = 500 µm, n = 3. b AChE co-localizes with HOPX+ outer radial glia in the oSVZ. Representative images of GW15 and GW22 prefrontal cortex at higher magnification show overlap of HOPX+ oRG and AChE+ cholinergic fibers. Arrowheads show co-expression of HOPX and AChE. Scale bar = 200 µm and 100 µm, n = 3. Abbreviations: A-P (anterior-posterior), ventricular zone (VZ), inner subventricular zone (iSVZ), outer subventricular zone (oSVZ) and Cortical plate (CP).

Fig. 2. Expression of Cholinergic receptor subunits CHRNA7 and CHRFAM7A in the developing cortex and cortical organoids.

a, a’, a” CHRNA7 and CHRFM7A RNA expression. a RNAscope for CHRNA7, and a’ BaseScope for CHRFAM7A, with coimmunostaining of radial glia marker SOX2 shows expression of both subunits in progenitor cells at GW17 (white arrowheads). CHRFAM7A RNA is enriched along the apical endfeet. b, b’ Immunostaining of CHRNA7 and SOX2, in VZ, oSVZ and CP, shown in GW14, GW18, and GW23 cortical samples c. Human-specific CHRFAM7A protein is enriched in the VZ. Immunostaining of CHRFAM7A validates enrichment of CHRFAM7A in the apical endfeet of the developing cortex. Colocalization of CHRFAM7A protein with SOX2 is observed in the VZ and oSVZ (white arrows). d, d’, e CHRFAM7A protein is expressed in ventricular RG in cerebral organoids. Immunostaining of CHRFAM7A in cortical organoids at weeks 7–9 and weeks 13–15 replicate primary tissue with expression of CHRFAM7A in SOX2+ progenitors and enrichment within neural rosettes. Abbreviations: ventricular zone (VZ), inner subventricular zone (iSVZ), outer subventricular zone (oSVZ) and Cortical plate (CP), n = 3.

Fig. 3. Activation of cholinergic signaling in primary cortical organotypic slices by agonist treatment increases progenitor cells and inhibits neuronal differentiation.

a Scheme showing treatment of cortical organotypic slices with broad (ACh) and specific (nicotine, PNU) agonists for 5 consecutive days. EdU was added on Day 4 and slices fixed with 4% PFA on Day 5 for Immunohistochemistry (IHC). b Representative images showing GW18 organotypic slices post ACh and nicotine treatment, immunostained for SOX2 + , KI67 + , EdU + , and co-stained with DAPI. Scale bar = 100µm. c Quantification of SOX2, KI67, EdU, and EOMES over total DAPI, post broad agonist treatment, n = 3. Upon ACh or nicotine treatment, the number of SOX2+ progenitors increased significantly to 42.1% (p = 1.12E-06), and 38.1% (p = 0.0007) of DAPI+ cells respectively, compared to 30.8% in untreated (UT) control. KI67+ proliferating cells increased to 23.3% (ACh, p = 1.61E-06) and 22.1% (nicotine, p = 2.66E.05), compared to 15.5% in controls. We observed an increase in EdU+ cells (24% for ACh, p = 8.61E-05; 21.4% for nicotine, p = 0.016; 18% UT control). d Representative immunostaining for EOMES and NEUN in organotypic slices treated with agonists. Scale bar = 100 µm. EOMES+ intermediate progenitor cells (IPCs) increased post nicotine to 18.2% (p = 5.47E-05) from 13.2%, while remaining unchanged after ACh. Quantification for NEUN and CTIP2 shows changes in neuronal proportions post agonist treatment. NEUN+ neurons were reduced to 64.3% with ACh (p = 0.02) and reduced to 43.5% (p = 2.93E-09) with nicotine, compared to 73.4% in the UT control. Deep layer CTIP2+ neurons in the IZ were reduced from 6.3% to 3.9% (p = 1.25E-05) with ACh, and 2.9% (p = 2.14E-08) with nicotine. e Immunostaining of SOX2, KI67, and EdU post PNU treatment. Scale bar = 100 µm. f Quantification for markers post specific activation of α7 nAChR. PNU increased SOX2+ cells to 44% compared to 39% in the DMSO control, but did not reach statistical significance (p = 0.054). KI67+ cells increased to 24.2% (p = 0.003) from 20.5%. PNU did not significantly impact EdU incorporation. NEUN+ neurons reduced from 63.6% in DMSO control to 45.5% (p = 0.001) with PNU, (n = 3), Source Data, two-sided t-test, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001. ns not significant.

Fig. 4. Activation of cholinergic signaling in cortical organoids shows comparable trends to primary tissue.

a Scheme of cortical organoid differentiation using the Modified Sasai Protocol, followed by treatment with broad (ACh) and specific (nicotine, PNU) agonists. Three control cortical organoid lines were treated at differentiation Week 7–8, for 5 consecutive days, PFA fixed on Day 5 and processed for IHC. b Representative images of cortical organoids, immunostained for SOX2, CTIP2 and SATB2 co-stained with DAPI. Scale bar = 100 µm. c Representative images of cortical organoids immunostained for NEUN and co-stained with DAPI. Scale bar = 100 µm. d Quantification for SOX2, NEUN, CTIP2 and SATB2. SOX2+ cells in ACh and nicotine increased to 37% (p = 0.01) and 34% (p = 0.001), respectively, compared to 27.4% in UT controls. The number of NEUN+ neurons was reduced from 52% to 34% (p = 0.002) in ACh and 40.4% (p = 0.02) in nicotine. SATB2+ neurons changed from 8.5% in control to 1.5% (p = 0.007) and 1.4% (p = 0.007) with ACh and nicotine treatment, respectively. e Representative images of cortical organoids post treatment with specific agonist, PNU and DMSO control stained for SOX2, NEUN, and DAPI. Scale bar = 100 µm. f Quantifications for SOX2 and NEUN post PNU treatment. In PNU treated organoids, SOX2+ progenitors increased from 29% in DMSO to 35% (p = 0.07) in PNU-treated conditions. NEUN+ cells were reduced to 40% (p = 0.02) in PNU treated cortical organoids, compared to 52% in DMSO controls, n = 3, Source Data, two-sided t-test, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001. ns not significant.

Fig. 5. Loss-of-function of CHRNA7 and CHRFAM7A receptor subunits shows opposite phenotypic changes compared to cholinergic activation.

a Experimental design for organotypic slice culture preparation and treatment with lentiviral mediated-shRNAs-directed against CHRNA7 or CHRFAM7A, and a scrambled control. Samples were fixed after 7-days with 4% PFA and processed for IHC. b Tile scan of example shCHRNA7 sample highlighting the regions used for imaging and quantifications c. Representative images of lentiviral infected, RFP+ cells, co-immunostained with NEUN+ (arrows) from GW21 with shCONTROL, shCHRNA7 and shCHRFAM7A. Scale bar = 50 µm. Representative images of lentiviral infected, RFP+ shCONTROL, shCHRNA7 and shCHRFAM7A cells, co-immunostained with SOX2, S100B, OLIG2 and NEUROD1 (arrowheads) at GW15 and GW17. Scale bar = 50 µm and 100 µm. d Quantification of SOX2, KI67, and EOMES upon knockdown of nAChRs. SOX2+ progenitors were reduced from 25% in shCONTROL to 15.4% in shCHRNA7 (p = 0.07) and 14% in shCHRFAM7A (p = 0.009). KI67+ dividing progenitors were also reduced, with 8.3% in the presence of shCHRNA7 (p = 0.03) and 7.1% with shCHRFAM7A (p = 0.01), compared to 17% in the shCONTROL. e Quantification of NEUROD1+ immature neurons upon CHRFAM7A knockdown increased from 15.3% in shCONTROL to 30% in shCHRFAM7A (p = 0.0032). NEUN+ neurons upon nAChR knockdown showed the number of NEUN+ neurons increased from 22.5% in shCONTROL to 52% upon knockdown of CHRNA7 (p = 0.009), and to 53% upon knockdown of CHRFAM7A (p = 0.01). f Quantification of S100B+ astrocytes upon AChR knockdown reduced from 11 to 6% in shCHRNA7 (p = 0.057) and to 5.5% in shCHRFAM7A (p = 0.05). Quantification of OLIG2+ oligodendrocytes upon AChR knockdown reduced from 12% to 6% in shCHRNA7 (p = 0.04) and to 3% in shCHRFAM7A (p = 0.01), with n = 3. Source Data, two-sided t-test, *p = 0.05, **p = 0.01, ns not significant.

Fig. 6. Nicotine exposure and loss-of-function of AChRs alters Hippo signaling effectors.

a Workflow showing nicotine exposure and nAChR knockdown in primary dissociated cortical cell cultures. Cells were fixed on day 5 following the Parse Biosciences Fixation protocol and processed for Split-sequencing, a combinatorial barcoding approach where different barcodes are added in each step. We obtained 8 sublibraries. Gene expression libraries were prepared and sequenced using Illumina NovaSeq 2 Flowcell. b UMAP clustering of single-cell RNA sequencing (scRNAseq) data identifies major expected cell types. c scRNAseq analysis reveals anticipated patterns of marker gene expression across clusters. SOX2, PAX6, VIM, NES are expressed in radial glia and oRG clusters, while TUBB, RBFOX3, TBR1, BCL11B and SATB2 are expressed by neuronal clusters. d Genes up- and downregulated upon nicotine exposure in dividing neurogenic RG cells. e Genes up- and downregulated upon nicotine exposure in immature neurons. One of the top downregulated genes is DCHS1, a canonical ligand of Hippo signaling. f Gene Ontology (GO) for dividing neurogenic RG identifies significant pathways and processes upregulated post nicotine exposure, including Semaphorin-plexin and homophilic cell adhesion signaling. g Genes up- and downregulated following knockdown of CHRNA7 in oRGs identifiies Hippo signaling receptor FAT3 among upregulated genes. h Knockdown of CHRFAM7A in oRGs also identifies Hippo signaling receptor FAT3 among upregulated genes. All DEGs are listed in Source Data.

Fig. 7. Hippo signaling effector YAP1 is downstream of cholinergic signaling.

a Scheme showing the ACh, NIC exposure and nAChR knockdown in primary dissociated neural cultures, derived from whole cortices. Cells were treated from Day 0 to Day 5 and protein lysates were prepared RIPA buffer. b Western blotting for Hippo co-activator YAP1 showed an upregulation with NIC treatment and a downregulation with KD of CHRNA7 and CHRFAM7A, compared to respective control samples. B-ACTIN was used as a loading control. c In-silico analysis of published single-cell sequencing data shows the RNA expression of several known Hippo effectors in relevant neuronal cell types. YAP1, FAT3, CRB2 and DCHS1 are highly expressed in RGs and oRGs. d Representative images showing protein expression of YAP1, DCHS1, SOX2 and EOMES in primary cortical tissue at GW23, n = 3. e Scheme showing the rescue experiment, where cortical organotypic slices were either treated with broad (ACh, NIC) agonists or a YAP1 inhibitor (TAT- PDHPS1) or a combination of both, for 5 consecutive days. Samples were fixed with 4% PFA and processed for immunofluorescence. f Immunostaining shows upregulation of YAP1 protein expression and increased nuclear translocation (arrows) post agonist treatment in oSVZ, compared to untreated controls. TAT, Acetylcholine+TAT and Nicotine+TAT show relatively similar YAP1 expression and localization as the untreated control, n = 3. g Quantifications post ACh, NIC, TAT, ACh+TAT and NIC + TAT samples compared to untreated controls show full rescue of HOPX+ cells over total DAPI in ACh+TAT-treated, and partial rescue in NIC + TAT-treated samples (Source Data), two-sided t-test, *p = 0.05, **p = 0.01, ns not significant, n = 3. h Representative immunostaining for RFP, YAP1 and DAPI show downregulation of YAP1 protein in RFP+ cells in shCHRNA7 and shCHRFAM7A samples (arrows), compared to RFP+ cells in shCONTROL. (All scale bars are mentioned on the image panels).