The human-specific duplicated α7 gene inhibits the ancestral α7, negatively regulating nicotinic acetylcholine receptor-mediated transmitter release
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100341
ISSN1083-351X
AutoresCarolina Martín-Sánchez, Eva Alés, Santiago Balseiro-Gómez, Gema Atienza, Francisco Arnalich, Anna Bordas, José Luis Cedillo, María Rueda-Extremera, Arturo Chávez‐Reyes, Carmen Montiel,
Tópico(s)Ion channel regulation and function
ResumoGene 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. 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. The α7 nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in neurons and nonneuronal cells of the human brain where it mostly forms homopentameric receptors composed of five α7 subunits (1Albuquerque E.X. Pereira E.F. Alkondon M. Rogers S.W. Mammalian nicotinic acetylcholine receptors: From structure to function.Physiol. Rev. 2009; 89: 73-120Crossref PubMed Scopus (1173) Google Scholar, 2Fasoli F. Gotti C. Structure of neuronal nicotinic receptors.Curr. Top. Behav. Neurosci. 2015; 23: 1-17Crossref PubMed Scopus (28) Google Scholar). This nAChR subtype is widely distributed in the central nervous system (CNS), although its expression is particularly prominent in the hippocampus and prefrontal cortex, two key regions involved in neurocognitive function (see Ref. 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Recently our group identified a possible endogenous candidate that may interfere with α7-nAChR function, the CHRFAM7A chimeric gene, which is evolutionarily a relatively recent gene since it appears in the human genome after its divergence from other higher primates (30Locke D.P. Archidiacono N. Misceo D. Cardone M.F. Deschamps S. Roe B. Rocchi M. Eichler E.E. Refinement of a chimpanzee pericentric inversion breakpoint to a segmental duplication cluster.Genome Biol. 2003; 4R50Crossref PubMed Google Scholar). The new hybrid gene results from partial duplication (exons 5–10) of the parent CHRNA7 gene, coding the α7 subunit that forms the α7-nAChR, fused to the FAM7A genetic element (31Gault J. Robinson M. Berger R. Drebing C. Logel J. Hopkins J. Moore T. Jacobs S. Meriwether J. Choi M.J. Kim E.J. Walton K. Buiting K. Davis A. 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McFadden W. Shin J.H. Deep-Soboslay A. Ye T. Li C. Kleinman J.E. Wang K.H. Lipska B.K. CHRNA7 and CHRFAM7A mRNAs: Co-localized and their expression levels altered in the postmortem dorsolateral prefrontal cortex in major psychiatric disorders.Am. J. Psychiatry. 2015; 172: 1122-1130Crossref PubMed Scopus (39) Google Scholar, 35Kalmady S.V. Agrawal R. Venugopal D. Shivakumar V. Amaresha A.C. Agarwal S.M. Subbanna M. Rajasekaran A. Narayanaswamy J.C. Debnath M. Venkatasubramanian G. CHRFAM7A gene expression in schizophrenia: Clinical correlates and the effect of antipsychotic treatment.J. Neural Transm. (Vienna). 2018; 125: 741-748Crossref PubMed Scopus (8) Google Scholar), the functional role of the chimeric gene was long unidentified until we reported that its product, the dupα7 subunit, acted as a dominant negative regulator of α7-nAChR-induced currents in a pioneering electrophysiological study conducted in Xenopus oocytes (36de Lucas-Cerrillo A.M. Maldifassi M.C. Arnalich F. Renart J. Atienza G. Serantes R. Cruces J. Sánchez-Pacheco A. Andrés-Mateos E. Montiel C. Function of partially duplicated human α7 nicotinic receptor subunit CHRFAM7A gene: Potential implications for the cholinergic anti-inflammatory response.J. Biol. Chem. 2011; 286: 594-606Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Our finding was corroborated shortly afterward by others, also in oocytes (37Araud T. Graw S. Berger R. Lee M. Neveu E. Bertrand D. Leonard S. The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7∗nAChR function.Biochem. Pharmacol. 2011; 82: 904-914Crossref PubMed Scopus (84) Google Scholar) and, a few years later, by our own group in diverse mammalian cell types. Thus, we reported that dupα7 overexpression inhibits, both in vitro and in vivo, α7-nAChR-mediated protumorigenic activity in human cell lines from non-small-cell lung cancer (NSCLC) (38Cedillo J.L. Bordas A. Arnalich F. Esteban-Rodríguez I. Martín-Sánchez C. Extremera M. Atienza G. Rios J.J. Arribas R.L. Montiel C. Anti-tumoral activity of the human-specific duplicated form of α7-nicotinic receptor subunit in tobacco-induced lung cancer progression.Lung Cancer. 2019; 128: 134-144Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Furthermore, using GH4C1 rat pituitary cells and RAW264.7 mouse macrophages transfected with epitope- or fluorescent protein-tagged α7 or dupα7 constructs, we identified the mechanism underlying dupα7 interference in α7-nAChR function. This mechanism consists of the physical interaction between dupα7 and α7 subunits generating heteromeric nAChRs that largely remain mainly trapped in the endoplasmic reticulum (39Maldifassi M.C. Martín-Sánchez C. Atienza G. Cedillo J.L. Arnalich F. Bordas A. Zafra F. Gimenez C. Extremera M. Renart J. Montiel C. Interaction of the α7-nicotinic subunit with its human-specific duplicated dupα7 isoform in mammalian cells: Relevance in human inflammatory responses.J. Biol. Chem. 2018; 293: 13874-13888Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Thus, the dupα7 sequestration of α7 subunits reduced membrane expression of functional homomeric α7-nAChRs, attenuating their recognized anti-inflammatory capacity in lipopolysaccharide-stimulated macrophages (39Maldifassi M.C. Martín-Sánchez C. Atienza G. Cedillo J.L. Arnalich F. Bordas A. Zafra F. Gimenez C. Extremera M. Renart J. Montiel C. Interaction of the α7-nicotinic subunit with its human-specific duplicated dupα7 isoform in mammalian cells: Relevance in human inflammatory responses.J. Biol. Chem. 2018; 293: 13874-13888Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The α7 subunit and its duplicate form are naturally coexpressed in the same human cell types, including neuronal, immune, or tumor cells (see Ref. (40Costantini T.W. Dang X. Coimbra R. Eliceiri B.P. Baird A. CHRFAM7A, a human-specific and partially duplicated α7-nicotinic acetylcholine receptor gene with the potential to specify a human-specific inflammatory response to injury.J. Leukoc. Biol. 2015; 97: 247-257Crossref PubMed Scopus (35) Google Scholar) and references there). Thus, it is to be expected that the dupα7 subunit would behave as an endogenous negative regulator of α7-nAChR-mediated activity in neurons, just as it does in macrophages or tumor cells (38Cedillo J.L. Bordas A. Arnalich F. Esteban-Rodríguez I. Martín-Sánchez C. Extremera M. Atienza G. Rios J.J. Arribas R.L. Montiel C. Anti-tumoral activity of the human-specific duplicated form of α7-nicotinic receptor subunit in tobacco-induced lung cancer progression.Lung Cancer. 2019; 128: 134-144Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 39Maldifassi M.C. Martín-Sánchez C. Atienza G. Cedillo J.L. Arnalich F. Bordas A. Zafra F. Gimenez C. Extremera M. Renart J. Montiel C. Interaction of the α7-nicotinic subunit with its human-specific duplicated dupα7 isoform in mammalian cells: Relevance in human inflammatory responses.J. Biol. Chem. 2018; 293: 13874-13888Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). This last hypothesis, still unexplored, is the one addressed here using the SH-SY5Y human neuroblastoma cell line to assess: (i) the physical interaction between α7 and dupα7 subunits in fluorescent protein-tagged α7/dupα7 transfected cells; and (ii) the functional impact of the above interaction in cell variants with different dupα7 expression levels. Immunoblotting, quantitative real-time polymerase chain reaction (RT-PCR), Föster resonance energy transfer (FRET) analysis combined with functional assays of α7-nAChR-activity, in either single cells or cell populations of the above cell variants, allowed us to establish that the dupα7 subunit negatively regulates α7-nAChR-mediated control of exocytotic neurotransmitter release in neuronal cell types. In order to avoid differences in the expression levels of dupα7 mRNA among different SH-SY5Y cultures transiently transfected with the dupα7.pcDNA3.1/Myc-His construct, we stably transfected the cells with this construct, or with its corresponding empty vector pcDNA3.1/Myc-His, as described in the corresponding Methods section. A total of seven positive clones resistant to G418 obtained by lipofection (L1 and L2) or nucleofection (N1-N5) of dupα7-Myc were initially selected. In all of them, the dupα7 or α7 mRNA levels were analyzed by qPCR in order to select those clones that, together with the clear overexpression of dupα7, presented endogenous α7 mRNA levels similar to those found in nontransfected cells (Control). We found two clones (L1 and N1) that fit the above requirements (Fig. 1, panels A and B). Since dupα7 mRNA overexpression is much more pronounced in the N1 than in the L1 clones, we proceeded to determine if this difference was maintained at the protein level. Immunoblot data using the anti-Myc antibody to detect the foreign dupα7-Myc protein overexpressed in L1 and N1 clones show that there were no significant differences in protein expression levels (Fig. 1C), so both clones were assayed in parallel throughout our study. Using FRET confocal imaging analysis in SH-SY5Y cells cotransfected with two pairs of α7 and dupα7 constructs (α7-GFP:dupα7-Cherry or dupα7-GFP:α7-Cherry), we evaluated whether dupα7 could physically interact with α7 subunits, thus modulating the α7-nAChR-mediated control of neurotransmission. The results (Fig. 1D) provide evidence that both nAChR subunits were in sufficient proximity to interact with each other as part of a heteropentameric nAChR. Thus, the left panel of the figure shows representative confocal images acquired before (pre) and after (post) acceptor [α7-Cherry] photobleaching at 561 nm in the framed area; the increase in emission at 488 nm of the donor [dupα7-GFP (post)] in the cell analyzed after coexpression of the dupα7-GFP:α7-Cherry pair is worth noting. The right panel of the figure represents the scatter plots of individual data points corresponding to the FRET efficiency values, expressed as a percentage of the maximum efficiency, obtained in the region selected for acceptor photobleaching in cells transfected with the two pairs of constructs assayed in this study at a 1:1 ratio. The error bars represent mean ± standard deviation (SD). The significantly higher FRET efficiency values when dupα7 was the donor of the pair are also worth noting. Consequently, the subsequent experiments were designed to evaluate the functional consequences of this dupα7/α7 interaction, in both cell populations and single cells. One of the most distinctive functions of α7-nAChR in neurons is to promote the exocytotic release of several neurotransmitters as the result of the [Ca2+]i rise induced by receptor stimulation. Therefore, the next experiments aimed to evaluate whether overexpression of dupα7 in SH-SY5Y cells interfered with the [Ca2+]i response induced by 1-s pulses of increasing concentrations of PNU 282987 (1 nM up to 10 μM) applied to cell populations. To amplify the responses induced by the selective α7-nAChR agonist and thus facilitate the subsequent analysis of their possible modulation by dupα7, a fixed concentration of the positive allosteric modulator (PAM) of the α7-nAChR (PNU 120596; 0.5 μM) was added to the cell medium from 10 min before and during the α7-nAChR agonist pulse. Figure 2A shows the concentration–response curves to the agonist in nontransfected cells (Control), in both the absence and presence of the PAM; it can be observed that the last agent greatly enhanced the agonist-induced response at all tested concentrations. Figure 2B shows the original fluorescence traces induced by different concentrations of PNU 282987 (+PAM) in control cells (black traces; left panel) or in cells expanded from the N1 clone (red traces; right panel). Figure 2, C and D show pooled results from independent cultures (n = 4–12) of normalized [Ca2+]i responses (Δ[Ca2+]i) evoked by increasing concentrations of PNU 282987 in the four cell variants tested [control cells, and cells overexpressing dupα7 (clones L1 and N1), or empty vector]. The Δ[Ca2+]i signals were normalized as a percentage of the maximum response induced by PNU 282987 (3 μM; 100%) in control cells (Fig. 2C) or in the corresponding cell variant (Fig. 2D). The analysis of variance (ANOVA) applied to data in Figure 2C showed that while the Δ[Ca2+]i signal induced by PNU 282987 in cells overexpressing the empty vector was indistinguishable from that found in control cells, the overexpression of dupα7 significantly reduced the α7-nAChR-mediated signal, particularly at the highest agonist testing concentrations (from 30 nM to 10 μM), but not the signal generated by a depolarizing stimulus of high K+ (70 mM, 1 s). The table inserted in Figure 2D shows the EC50 and slope values obtained from the concentration–response curves of PNU 282987 in the four cell variants tested; the application of the above statistical analysis to both parameters did not show significant differences between the Δ[Ca2+]i signals generated in the four variants. To gain further insights into dupα7 negative regulation of the α7-nAChR-induced [Ca2+]i rise, we evaluated this regulation at the single cell level by fluorescence microscopy. The SH-SY5Y cells tested in these experiments belong to the next three cell variants: control, L1 clone, and N1 clone. Individual cells were stimulated with a 1-min pulse of PNU 282987 (1 μM) in the presence of PAM (1 μM) added to the perfusion medium 20 min before and during the application of the stimulus. After a washout period, the cells were exposed to a final high K+ pulse (100 mM, 30 s) to exclude from subsequent analyses those cells that had not responded to the depolarizing stimulus. Figure 3A shows original traces of the fluorescence increase (ΔF) induced by the above two pulses applied successively in two Fura 2-loaded cells representative of the Control (upper panel) and the L1 clone (lower panel) groups. Note that overexpression of dupα7 markedly reduces the α7-nAChR-mediated signal but not that induced by the depolarizing stimulus. In fact, the high K+-evoked response is higher in the dupα7 overexpressing cell than in the control cell, probably because the [Ca2+]i signal induced by PNU 282987 in the latter cell had not yet returned to the basal level when the depolarizing stimulus was applied. Figure 3B shows pooled time-course results of the [Ca2+]i signal evoked by PNU 282987 in 3 to 4 individual cells belonging to each tested cell variant; the signals recorded in the clones were normalized with respect to the signal obtained in control cells. Blockade of the α7-nAChR-induced signal was clearly seen in L1 and N1 clones with respect to control cells, but there were no significant differences in the blocked signal between the two types of clones. Figure 3C shows scatter plots of individual data points and statistical analyses of different kinetic parameters relative to the [Ca2+]i signal induced by PNU 282987 or high K+ in single cells from the three variants (control, clone L1, and clone N1) from independent cultures. The error bars represent mean ± SD of the values obtained in the number of cells appearing in parentheses. The overexpression of dupα7 significantly reduced the "Peak Amplitude" and the "Area Under the Curve" of the [Ca2+]i signal induced by α7-nAChR but not the signal evoked by the depolarizing stimulus. None of the other analyzed kinetic parameters, independently of the stimulus, was significantly affected by dupα7 overexpression. The [Ca2+]i rise induced by α7-nAChRs in neurons activates the SNARE protein complex and the consequent discharge of the vesicular content (neurotransmitter) into the synaptic space by an exocytotic mechanism. Thus, it is likely that the α7-nAChR-mediated exocytotic response in SH-SY5Y cells is affected by dupα7 overexpression, as is the [Ca2+]i signal induced by the same receptor subtype. The following experiments were aimed at evaluating this hypothesis at the single cell level. For this, synaptic vesicles of SH-SY5Y cells belonging to the three cell variants tested in this study (control, clone L1, and clone N1) were loaded with the fluorescent FM1-43 dye as described in the corresponding Methods section. Subsequently, the exocytosis of the labeled vesicles was promoted by two successive 1-min pulses of 1 μM PNU 282987 (+PAM, 1 μM) and high K+ (100 mM), separated by a washing period. Figure 4A shows two original destaining traces (loss of the fluorescent signal) indicative of the extent of the exocytotic response induced by the two stimuli in a Control cell and in a cell overexpressing dupα7 (Clone L1). The upper diagram of both registers illustrates the two key steps of what happened with the labeled vesicles in each cell type in response to the two stimuli: 1) PNU 282987 induced vesicular exocytosis (drop in the fluorescent signal) in the Control cell but not in the one with dupα7 overexpression; and 2), in contrast, high K+ was not able
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