The human-specific duplicated α7 gene inhibits the ancestral α7, negatively regulating nicotinic acetylcholine receptor-mediated transmitter release

Abstract

Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca²⁺]_i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.

Keywords: CHRFAM7A; calcium intracellular release; central nervous system; dupα7 nicotinic subunit; human genetics; human-specific duplicate genes; neurotransmitter release; α7 nicotinic acetylcholine receptors.

Figure 1

Expression levels of dupα7 and endogenous α7 subunits in control SH-SY5Y cells or in cells with stable dupα7-myc overexpression (Clones).A and B, normalized expression of the mRNAs of both nicotinic subunits determined by qPCR in control cells (value = 1) and in the clones obtained after lipofection (L1 and L2) or nucleofection (N1–N5). The solid circles overlaid on the bar graphs represent individual data points obtained in independent cultures for each condition. The error bars show mean ± SD. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 after comparison with the control. The two clones (L1 and N1) selected for the subsequent functional study are indicated by the black arrow. C, expression level of the foreign dupα7-myc protein, determined by immunoblot with the anti-myc antibody, in control cells or in the selected clones. At the top, a typical immunoblot; at the bottom, the protein expression values normalized with respect to β-actin expression. The error bars show mean ± SD from three independent cultures and solid circles the individual values for each condition. ^∗∗∗p < 0.001 compared with control. D, physical interaction between α7 and dupα7 subunits in SH-SY5Y cells evaluated by FRET efficiency analysis. Cells were transfected with the construct pairs α7-GFP:dupα7-Cherry or dupα7-GFP:α7-Cherry, at a ratio of 1:1. On the left, representative confocal images of FRET analysis performed in the selected area of a cell cotransfected with dupα7-GFP:α7-Cherry, where the fluorescence emitted by dupα7-GFP in the framed area is shown before (pre) and after (post) α7-Cherry photobleaching; scale bar 10 μm. On the right, scatter plots (mean ± SD) representing individual data points of FRET efficiency values (expressed as a percentage) determined in the number of cells analyzed (in parentheses) from three independent cultures. ^∗∗p < 0.01 after comparing the indicated values.

Figure 2

Effect of dupα7 overexpression on the α7-nAChR-mediated [Ca²⁺]_isignal in populations of SH-SY5Y cells.A, [Ca²⁺]_i signal induced by increasing concentrations of the selective α7-nAChR agonist, PNU 282987 (1 nM–10 μM), in the absence or presence of PNU 120596 (0.5 μM), a positive allosteric modulator (PAM) of the receptor. Peak fluorescence values induced by the agonist in both experimental conditions, obtained in triplicate, were expressed as a percentage of F_max − F_min (mean ± S.E.M.) and correspond to four independent cell cultures. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 after comparing the same concentration of the agonist in both curves. B, original traces of fluorescence intensity induced by increasing concentrations of PNU 282987 (+PAM) in control cells (black traces; left panel) or in cells expanded from the N1 clone (red traces; right panel). The signal was monitored for 35 s and the PNU 282987 (agonist) was added after measuring basal fluorescence for 5 s, as is indicated by the arrow. C and D, pooled results of the normalized [Ca²⁺]_i responses (Δ[Ca²⁺]_i) evoked by increasing concentrations of PNU 282987 (+PAM) in the four cell variants tested [control cells, clones L1 and N1 or transfected with empty vector]; finally, a depolarizing stimulus of high K⁺ (70 mM; 1 s) was applied at the end of the experiment. The Δ[Ca²⁺]_i signals were normalized as a percentage of the maximum response induced by PNU 282987 (3 μM; 100%) in control cells (panel C) or in the corresponding cell variant (panel D). Values are mean ± S.E.M. from the number of independent cultures indicated in parentheses for each cell variant in panel C. Table inserted (at the right of panel D) shows the EC₅₀ and Slope values obtained from the concentration–response curves of PNU 282987 in the four cell variants tested.

Figure 3

Analysis of various kinetic parameters related to the [Ca²⁺]_i signal generated by PNU 282987 or high K⁺ in single control or dupα7-overexpressing SH-SY5Y cells.A, original traces of fluorescence signal (ΔF_Ratio) induced by two successive pulses [1 μM PNU 282987 (+PAM, 1 μM) and 100 mM K⁺] applied 1 min apart to a Control cell or to a cell from Clone L1. B, time-course of the normalized [Ca²⁺]_i signal generated by the α7-nAChR agonist (+PAM) in individual cells from the three cell variants assayed (control and N1 and L1 clones). Each value represents mean ± S.E.M. of three cells for each variant. C, scatter plots of individual data points relative to different kinetic parameters obtained from the analysis of the [Ca²⁺]i signal evoked by PNU 282987 (+PAM) or high K⁺ in single cells corresponding to the three cell variants assayed. In parentheses, the number of cells from each variant. The error bars show mean ± SD. ^∗p < 0.05 and ^∗∗∗p < 0.001 after comparing the indicated values.

Figure 4

Effect of dupα7 overexpression on the exocytotic responses elicited by PNU 282987 and high K⁺ in single control or dupα7 overexpressing SH-SY5Y cells. Synaptic vesicles were loaded with the fluorescent FM1-43 membrane dye as described in the Method section. The cells were then thoroughly washed and subsequently subjected to two successive stimulations with 1 μM PNU 282987 (+PAM, 1 μM) and 100 mM K⁺ to induce fusion of labeled vesicles with the plasma membrane and subsequent exocytotic release of FM1-43, which is deduced by the loss of the fluorescent signal of the probe (destaining) in the aqueous solution. A, original traces of the fluorescent signal in response to both stimuli applied to a Control cell (left) or to a cell overexpressing dupα7 (Clone L1; right). In the upper part, diagram representing the FM1-43 dye used to evaluate the exocytotic responses to different stimuli in individual cells. B, timecourse of the fluorescent signal decay (destaining) reflecting the PNU 282987-induced exocytotic response in control cells or in cells from L1 and N1 clones; values represent mean ± S.E.M. from several cells (n = 3–4) for each cell variant. F₀ corresponds to the maximum fluorescence (100%) before the addition of the stimulus (black arrow). C, scatter plots of individual data points reflecting the percentage of the exocytotic response induced by PNU 28297 in single cells from the three cell variants; error bars show mean ± SD of the number of cells analyzed for each variant (in parentheses). ^∗p < 0.05 and ^∗∗∗p < 0.001 compared with control cells.

Figure 5

Silencing effectiveness of different siRNAs on dupα7 gene expression and its functional consequence on α7-nAChR-mediated neurotransmitter release in populations of SH-SY5Y cells. The efficacy of three different siRNAs designed for selectively silencing CHRFAM7A gene expression was double-checked by qPCR and immunoblot. A, bar graph showing mean ± SD of the normalized expression of dupα7 and α7 mRNAs in cells transfected with the tested siRNAs (1, 2 or 3); cells transfected with a commercial siRNA (control siRNA) and nontransfected cells were used as negative and positive controls, respectively. B, bar graph representing mean ± SD of the dupα7/β-actin or α7/β-actin protein ratio determined in the same population of cells assayed above; at the bottom, one original blot of a membrane cut above the 43 kDa molecular marker and incubated with the sc-5544 antibody to exclusively view the endogenous dupα7 subunit (≈41 kDa). Solid circles overlaid on the bar graphs in panels A and B represent individual data points obtained in independent cultures for each condition. ^∗p <0.05, ^∗∗p <0.01 and ^∗∗∗p <0.001 compared with non-transfected cells. C, original blot of a single membrane incubated with the sc-5544 antibody that recognizes two bands whose sizes correspond to the α7 (≈57 kDa) and dupα7 proteins. Only siRNA-1 and siRNA-3 showed selectivity and effectiveness in silencing dupα7 mRNA and protein expression, leaving the parent CHRNA7 gene expression unchanged. D, exocytotic dopamine (DA) release induced by 1 μM PNU 282987 (+PAM, 0.5 μM) or high K⁺ (100 mM) in populations belonging to three cell types: nontransfected, with stable dupα7 overexpression (clone N1), and with endogenous dupα7 silencing with siRNA-1. Net DA release, determined by ELISA, was normalized to the cell protein content. Solid circles overlaid on the bar graph represent individual data points obtained in 3 to 4 independent cultures for each condition. The error bars show mean ± SD. ^∗p < 0.05 and ^∗∗∗p < 0.001 after comparing the indicated values.